U.S. patent application number 13/255536 was filed with the patent office on 2012-02-16 for compounds for the treatment of metabolic disorders.
This patent application is currently assigned to PROSIDION LIMITED. Invention is credited to Oscar Barba, Susan Helen Davis, Matthew Colin Thor Fyfe, Revathy Perpetua Jeevaratnam, Karen Lesley Schofield, Thomas Staroske, Alan John William Stewart, Simon Andrew Swain, David Matthew Withall.
Application Number | 20120040953 13/255536 |
Document ID | / |
Family ID | 40600949 |
Filed Date | 2012-02-16 |
United States Patent
Application |
20120040953 |
Kind Code |
A1 |
Barba; Oscar ; et
al. |
February 16, 2012 |
Compounds for the Treatment of Metabolic Disorders
Abstract
The present invention is directed to therapeutic compounds which
have activity as agonists of GPR119 and are useful for the
treatment of metabolic disorders including type II diabetes.
Inventors: |
Barba; Oscar; (Oxford,
GB) ; Davis; Susan Helen; (Oxford, GB) ; Fyfe;
Matthew Colin Thor; (Oxford, GB) ; Jeevaratnam;
Revathy Perpetua; (Oxford, GB) ; Schofield; Karen
Lesley; (Oxford, GB) ; Staroske; Thomas;
(Oxford, GB) ; Stewart; Alan John William;
(Oxford, GB) ; Swain; Simon Andrew; (Oxford,
GB) ; Withall; David Matthew; (Oxford, GB) |
Assignee: |
PROSIDION LIMITED
Oxford
GB
|
Family ID: |
40600949 |
Appl. No.: |
13/255536 |
Filed: |
March 12, 2010 |
PCT Filed: |
March 12, 2010 |
PCT NO: |
PCT/GB2010/050442 |
371 Date: |
November 2, 2011 |
Current U.S.
Class: |
514/210.18 ;
514/252.03; 514/275; 514/316; 514/318; 544/238; 544/331; 546/187;
546/194 |
Current CPC
Class: |
A61P 3/00 20180101; A61P
3/10 20180101; A61P 9/12 20180101; A61P 43/00 20180101; A61P 3/08
20180101; A61P 3/06 20180101; A61P 3/04 20180101; C07D 413/14
20130101 |
Class at
Publication: |
514/210.18 ;
514/252.03; 514/275; 514/316; 514/318; 544/238; 544/331; 546/187;
546/194 |
International
Class: |
A61K 31/506 20060101
A61K031/506; A61K 31/4545 20060101 A61K031/4545; C07D 401/14
20060101 C07D401/14; A61P 3/06 20060101 A61P003/06; A61P 3/00
20060101 A61P003/00; A61P 3/04 20060101 A61P003/04; C07D 413/14
20060101 C07D413/14; A61K 31/501 20060101 A61K031/501; A61P 3/10
20060101 A61P003/10 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 12, 2009 |
GB |
0904287.0 |
Claims
1. A compound of formula (I) or a pharmaceutically acceptable salt
thereof: ##STR00120## wherein p is 1 or 2; when p is 2, Z is
N--C(O)OR.sup.4, N--C(O)NR.sup.4R.sup.5 or N-heteroaryl optionally
substituted by one or two groups selected from the group consisting
of C.sub.1-4 alkyl, C.sub.3-6 cycloalkyl optionally substituted by
C.sub.1-4 alkyl, C.sub.1-4 alkoxy, C.sub.1-4 haloalkyl and halogen;
when p is 1, Z can also be --N--CH.sub.2-phenyl wherein the phenyl
is optionally substituted by 1 or 2 groups independently selected
from the group consisting of C.sub.1-4 alkyl, C.sub.1-4 haloalkyl
and halo; A is a para-substituted phenyl or a para-substituted
6-membered heteroaryl ring containing one or two nitrogen atoms; B
is a 5-membered heteroaryl ring containing one of more heteroatoms
selected from the group consisting of N, O and S or a
para-substituted 6-membered heteroaryl ring containing one or two
nitrogens; when B is a 5-membered heteroaryl ring X is
--O--CR.sup.6H-- or --CR.sup.7H--O--CR.sup.6H--; and when B is a
6-membered heteroaryl ring X is --O-- or CR.sup.6H--O--; R.sup.1 is
hydrogen, halo, cyano, C.sub.1-4 alkyl or C.sub.1-4 haloalkyl; q is
1 or 2; R.sup.2 is ##STR00121## phenyl optionally substituted by
one or more halo groups, or pyridyl optionally substituted by one
or more halo or methyl groups; R.sup.3 is independently halo or
methyl; n is 0 or 1; m is 0, 1 or 2; R.sup.4 is C.sub.2-6 alkyl or
C.sub.3-6 cycloalkyl wherein the cycloalkyl is optionally
substituted by C.sub.1-4 alkyl; R.sup.5 is hydrogen or C.sub.1-4
alkyl; and R.sup.6 and R.sup.2 are independently hydrogen or
C.sub.1-2 alkyl.
2. A compound according to claim 1, or a pharmaceutically
acceptable salt thereof, having the stereochemistry as defined in
formula (Ia): ##STR00122##
3. A compound according to claim 1, or a pharmaceutically
acceptable salt thereof, wherein p is 2.
4. A compound according to claim 1, or a pharmaceutically
acceptable salt thereof, wherein Z is N--C(O)OR.sup.4.
5. A compound according to claim 4, or a pharmaceutically
acceptable salt thereof, wherein R.sup.4 is C.sub.2-6 alkyl.
6. A compound according to claim 1, or a pharmaceutically
acceptable salt thereof, wherein Z is N-heteroaryl optionally
substituted by one or two groups selected from the group consisting
of C.sub.1-4 alkyl, C.sub.3-6 cycloalkyl optionally substituted by
C.sub.1-4 alkyl, C.sub.1-4 alkoxy, C.sub.1-4 haloalkyl and
halogen.
7. A compound according to claim 6, or a pharmaceutically
acceptable salt thereof, wherein Z is optionally substituted
oxadiazole or pyrimidine.
8. A compound according to claim 1, or a pharmaceutically
acceptable salt thereof, wherein A is phenyl, pyridyl or
pyrimidinyl.
9. A compound according to claim 1, or a pharmaceutically
acceptable salt thereof, wherein R.sup.1 is hydrogen.
10. A compound according to claim 1, or a pharmaceutically
acceptable salt thereof, wherein B is oxadiazole, thiazole or
pyridine.
11. A compound according to claim 1, or a pharmaceutically
acceptable salt thereof, wherein when B is a 5-membered heteroaryl
ring X is -0-CR.sup.6H--, and when B is a 6-membered heteroaryl
ring X is CR.sup.6H--O--.
12. A compound according to claim 1, or a pharmaceutically
acceptable salt thereof, wherein R.sup.2 is phenyl substituted by
one or more halo groups.
13. A compound according to claim 12, or a pharmaceutically
acceptable salt thereof, wherein R.sup.2 is phenyl substituted by
one or more fluoro groups.
14. A compound according to claim 1, or a pharmaceutically
acceptable salt thereof, wherein R.sup.6 and R.sup.7 are
independently hydrogen or methyl.
15. (canceled)
16. A pharmaceutical composition comprising a compound according to
claim 1, or a pharmaceutically acceptable salt thereof; and a
pharmaceutically acceptable carrier.
17. A method for the treatment of a disease or condition in which
GPR119 plays a role, said method comprising administering to a
subject in need thereof an effective amount of a compound of claim
1, or a pharmaceutically acceptable salt thereof.
18. A method for the treatment of a disease or condition in which
GPR119 and DPP-IV play a role, said method comprising administering
to a subject in need thereof an effective amount of a compound
according to claim 1, or a pharmaceutically acceptable salt
thereof.
19. A method for the treatment of type II diabetes, said method
comprising administering to a subject in need thereof an effective
amount of a compound according to claim 1, or a pharmaceutically
acceptable salt thereof.
20. A method for the treatment of obesity, metabolic syndrome
(syndrome X), impaired glucose tolerance, hyperlipidemia,
hypertriglyceridemia, hypercholesterolemia, low HDL levels or
hypertension, said method comprising administering to a patient in
need thereof an effective amount of a compound according to claim
1, or a pharmaceutically acceptable salt thereof.
21. A compound according to claim 1 which is selected from the
group consisting of:
4-(5-{6-[(3R,4S)-3-Amino-4-(2-fluorophenyl)pyrrolidin-1-yl]pyridine-3-yl}-
-[1,2,4]oxadiazol-3-ylmethoxy)piperidine-1-carboxylic acid
isopropyl ester or a pharmaceutically acceptable salt thereof,
4-(5-{2-[(3R,4S)-3-Amino-4-(2-fluorophenyl)pyrrolidin-1-yl]pyrimidin-5-yl-
}-[1,2,4]oxadiazol-3-ylmethoxy)piperidine-1-carboxylic acid
isopropyl ester p-toluenesulfonic acid salt or a free base thereof,
4-(5-{2-[(3R,4S)-3-Amino-4-(2,5-difluorophenyl)pyrrolidin-1-yl]pyrimidin--
5-yl}-[1,2,4]oxadiazol-3-ylmethoxy)piperidine-1-carboxylic acid
isopropyl ester p-toluenesulfonic acid salt or a free base thereof,
4-(5-{2-[(3R,4S)-3-Amino-4-(2,4-difluorophenyl)pyrrolidin-1-yl]pyrimidin--
5-yl}-[1,2,4]oxadiazol-3-ylmethoxy)piperidine-1-carboxylic acid
isopropyl ester p-toluenesulfonic acid salt or a free base thereof,
4-(3-{6-[(3R,4S)-3-Amino-4-(2-fluorophenyl)pyrrolidin-1-yl]pyridine-3-yl}-
-[1,2,4]oxadiazol-5-ylmethoxy)piperidine-1-carboxylic acid
isopropyl ester p-toluenesulfonic acid salt or a free base thereof,
4-(3-{6-[(3S,4R)-3-Amino-4-(2-fluorophenyl)pyrrolidin-1-yl]pyridine-3-yl}-
-[1,2,4]oxadiazol-5-ylmethoxy)piperidine-1-carboxylic acid
isopropyl ester p-toluenesulfonic acid salt or a free base thereof,
4-(3-{6-[(3S,4R)-3-Amino-4-(2-fluorophenyl)pyrrolidin-1-yl]pyridine-3-yl}-
-[1,2,4]oxadiazol-5-ylmethoxy)piperidine-1-carboxylic acid
isopropyl ester p-toluenesulfonic acid salt or a free base thereof,
4-(3-{6-[(3R,4S)-3-Amino-4-(2,5-difluorophenyl)pyrrolidin-1-yl]pyridin-3--
yl}-[1,2,4]oxadiazol-5-ylmethoxy)piperidine-1-carboxylic acid
isopropyl ester p-toluenesulfonic acid salt or a free base thereof,
4-[(R)-1-(3-{6-[(3R,4S)-3-Amino-4-(2,5-difluorophenyl)pyrrolidin-1-yl]pyr-
idin-3-yl}-[1,2,4]oxadiazol-5-yl)ethoxy]piperidine-1-carboxylic
acid isopropyl ester p-toluenesulfonic acid salt or a free base
thereof,
4-[(R)-1-(5-{2-[(3R,4S)-3-Amino-4-(2,5-difluorophenyl)pyrrolidin-1-yl]pyr-
imidin-5-yl}-[1,2,4]oxadiazol-3-yl)ethoxy]piperidine-1-carboxylic
acid isopropyl ester or a pharmaceutically acceptable salt thereof,
4-[(R)-1-(5-{5-[(3R,4S)-3-Amino-4-(2,5-difluorophenyl)pyrrolidin-1-yl]pyr-
azin-2-yl}-[1,2,4]oxadiazol-3-yl)ethoxy]piperidine-1-carboxylic
acid isopropyl ester or a pharmaceutically acceptable salt thereof,
4-{5-[(3S,4R)-3-Amino-4-(2,5-difluorophenyl)-3,4,5,6-tetrahydro-2H-[1,2]b-
ipyridinyl-5'yl]-[1,2,4]oxadiazol-3-ylmethoxy}piperidine-1-carboxylic
acid isopropyl ester hydrochloride or a free base thereof,
4-{3-[(3R,4R)-3-Amino-4-(2,5-difluorophenyl)-3,4,5,6-tetrahydro-2H-[1,2]b-
ipyridinyl-5'-yl]-[1,2,4]oxadiazol-5-ylmethoxy}piperidine-1-carboxylic
acid isopropyl ester dihydrochloride or a free base thereof,
4-(3-{2-[(3R,4S)-3-Amino-4-(2,4-difluorophenyl)pyrrolidin-1-yl]pyrimidin--
5-yl}-[1,2,4]oxadiazol-5-ylmethoxy)piperidine-1-carboxylic acid
isopropyl ester p-toluenesulfonic acid salt or a free base thereof,
4-(3-{2-[(3R,4S)-3-Amino-4-(2,5-difluorophenyl)piperidin-1-yl]pyrimidin-5-
-yl}-[1,2,4]oxadiazol-5-ylmethoxy)piperidine-1-carboxylic acid
isopropyl ester p-toluenesulfonic acid salt or a free base thereof,
4-[(R)-1-(3-{2-[(3R,4S)-3-Amino-4-(2,4-difluorophenyl)pyrrolidin-1-yl]pyr-
imidin-5-yl}-[1,2,4]oxadiazol-5-yl)ethoxy]piperidine-1-carboxylic
acid isopropyl ester p-toluenesulfonic acid salt or a free base
thereof,
4-[(R)-1-(3-{2-[(3R,4S)-3-Amino-4-(2,5-difluorophenyl)pyrrolidin-1-yl]pyr-
imidin-5-yl}-[1,2,4]oxadiazol-5-yl)ethoxy]piperidine-1-carboxylic
acid isopropyl ester p-toluenesulfonic acid salt or a free base
thereof,
4-(3-{2-[(3R,4S)-3-Amino-4-(2-fluorophenyl)pyrrolidin-1-yl]pyrimidin-5-yl-
}-[1,2,4]oxadiazol-5-ylmethoxy)piperidine-1-carboxylic acid
isopropyl ester p-toluenesulfonic acid salt or a free base thereof,
4-[(R)-1-(3-{2-[(3R,4S)-3-Amino-4-(2-fluorophenyl)pyrrolidin-1-yl]pyrimid-
in-5-yl}-[1,2,4]oxadiazol-5-yl)ethoxy]piperidine-1-carboxylic acid
isopropyl ester p-toluenesulfonic acid salt or a free base thereof,
4-[(R)-1-(5-{6-[(3R,4S)-3-Amino-4-(2,5-difluorophenyl)pyrrolidin-1-yl]pyr-
idazin-3-yl}-[1,2,4]oxadiazol-3-yl)ethoxy]piperidine-1-carboxylic
acid isopropyl ester or a pharmaceutically acceptable salt thereof,
3-[(R)-1-(3-{2-[(3R,4S)-3-Amino-4-(2,5-difluorophenyl)pyrrolidin-1-yl]pyr-
imidin-5-yl}-[1,2,4]oxadiazol-5-yl)ethoxy]azetidine-1-carboxylic
acid isopropyl ester p-toluenesulfonic acid salt or a free base
thereof,
4-[(R)-1-(3-{5-[(3R,4S)-3-Amino-4-(2,5-difluorophenyl)pyrrolidin-1-yl]pyr-
azin-2-yl}-[1,2,4]oxadiazol-5-yl)ethoxy]piperidine-1-carboxylic
acid isopropyl ester or a pharmaceutically acceptable salt thereof,
4-[(R)-1-(5-{2-[(3R,4S)-3-Amino-4-(2,4,5-trifluorophenyl)pyrrolidin-1-yl]-
pyrimidin-5-yl}-[1,2,4]oxadiazol-3-yl)ethoxy]piperidine-1-carboxylic
acid isopropyl ester or a pharmaceutically acceptable salt thereof,
(R)-3-[(R)-1-(5-{2-[(3R,4S)-3-Amino-4-(2,5-difluorophenyl)pyrrolidin-1-yl-
]pyrimidin-5-yl}-[1,2,4]oxadiazol-3-yl)ethoxy]pyrrolidine-1-carboxylic
acid isopropyl ester or a pharmaceutically acceptable salt thereof,
(S)-3-[(R)-1-(5-{2-[(3R,4S)-3-Amino-4-(2,5-difluorophenyl)pyrrolidin-1-yl-
]pyrimidin-5-yl}-[1,2,4]oxadiazol-3-yl)ethoxy]pyrrolidine-1-carboxylic
acid isopropyl ester or a pharmaceutically acceptable salt thereof,
4-[(S)-1-(5-{2-[(3S,4S)-3-Amino-4-(2-oxopiperidin-1-yl)pyrrolidin-1-yl]py-
rimidin-5-yl}pyridin-2-yloxy)ethyl]piperidine-1-carboxylic acid
isopropyl ester or a pharmaceutically acceptable salt thereof,
4-[(S)-1-(5-{2-[(3R,4S)-3-Amino-4-(2,5-difluorophenyl)pyrrolidin-1-yl]pyr-
imidin-5-yl}pyridin-2-yloxy)ethyl]piperidine-1-carboxylic acid
isopropyl ester or a pharmaceutically acceptable salt thereof, and
4-[(R)-1-(5-{2-[(3R,4S)-3-Amino-4-(2,5-difluorophenyl)pyrrolidin-1-yl]pyr-
imidin-5-yl}pyridin-2-yloxy)ethyl]piperidine-1-carboxylic acid
isopropyl ester or a pharmaceutically acceptable salt thereof.
Description
BACKGROUND OF THE INVENTION
[0001] The present invention is directed to therapeutic compounds
useful for the treatment of metabolic disorders including type II
diabetes. In particular, the present invention is directed to
compounds which have activity as agonists of GPR119.
[0002] Drugs aimed at the pathophysiology associated with
non-insulin dependent type II diabetes have many potential side
effects and do not adequately address the dyslipidaemia and
hyperglycaemia in a high proportion of patients. Treatment is often
focused at individual patient needs using diet, exercise,
hypoglycaemic agents and insulin, but there is a continuing need
for novel antidiabetic agents, particularly ones that may be better
tolerated with fewer adverse effects.
[0003] Similarly, metabolic syndrome (syndrome X) places people at
high risk of coronary artery disease, and is characterized by a
cluster of risk factors including central obesity (excessive fat
tissue in the abdominal region), glucose intolerance, high
triglycerides and low HDL cholesterol, and high blood pressure.
Myocardial ischemia and microvascular disease is an established
morbidity associated with untreated or poorly controlled metabolic
syndrome.
[0004] Obesity is characterized by an excessive adipose tissue mass
relative to body size. Clinically, body fat mass is estimated by
the body mass index (BMI; weight(kg)/height(m).sup.2), or waist
circumference. Individuals are considered obese when the BMI is
greater than 30 and there are established medical consequences of
being overweight. It has been an accepted medical view for some
time that an increased body weight, especially as a result of
abdominal body fat, is associated with an increased risk for
diabetes, hypertension, heart disease, and numerous other health
complications, such as arthritis, stroke, gallbladder disease,
muscular and respiratory problems, back pain and even certain
cancers.
[0005] There is a continuing need for novel antidiabetic agents,
particularly ones that are well tolerated with few adverse effects
and in particular for agents which are weight neutral or preferably
cause weight loss.
[0006] GPR119 (previously referred to as GPR116) is a GPCR
identified as SNORF25 in WO00/50562 which discloses both the human
and rat receptors, U.S. Pat. No. 6,468,756 also discloses the mouse
receptor (accession numbers: AAN95194 (human), AAN95195 (rat) and
ANN95196 (mouse)).
[0007] In humans, GPR119 is expressed in the pancreas, small
intestine, colon and adipose tissue. The expression profile of the
human GPR119 receptor indicates its potential utility as a target
for the treatment of diabetes.
[0008] GPR119 agonists have been shown to stimulate the release of
GLP-1 from the GI tract. In doing so, GPR119 agonists (1) enhance
glucose-dependent insulin release from the pancreas leading to
improvements in oral glucose tolerance; (2) attenuate disease
progression by increasing .beta.-cell cAMP concentrations; and (3)
induce weight loss possibly through GLP-1's ability to reduce food
intake.
[0009] International Patent Applications WO2005/061489,
WO2006/070208, WO2006/067532, WO2006/067531, WO2007/003960,
WO2007/003961, WO2007/003962, WO2007/003964, WO2007/116229,
WO2007/116230, WO2007/138362, WO2008/081204, WO2008/081205,
WO2008/081206, WO2008/081207, WO2008/081208, WO2009/050522,
WO2009/050971, WO2010/004343, WO2010/004344, WO2010/004345,
WO2010/004347 and WO2010/00166 disclose GPR119 receptor
agonists.
[0010] Dipeptidyl peptidase IV (DPP-IV) is a ubiquitous, yet highly
specific, serine protease that cleaves N-terminal dipeptides from
polypeptides with L-proline or L-alanine at the penultimate
position. Studies with DPP-IV inhibitors show the principle role of
DPP-IV is in the inactivation GLP-1. By extending the duration of
action of GLP-1, insulin secretion is stimulated, glucagon release
inhibited, and gastric emptying slowed. DPP-IV inhibitors are of
use for the treatment of type II diabetes, examples of DPP-1V
inhibitors include vildagliptin, sitagliptin, alogliptin and
saxagliptin.
[0011] The possibility of using a combination of a GPR119 agonist
and a DPP-IV inhibitor has been suggested, however this requires
the administration of two separately formulated products to the
patient or the co-formulation of two active ingredients with the
inherent problems of achieving compatibility in the
physicochemical, pharmacokinetic and pharmacodynamic properties of
the two active ingredients. International Patent Application
WO2009/034388, published after the priority date of the present
application, discloses compounds having dual activity as agonists
of GPR119 and inhibitors of DPP-IV.
[0012] The compounds of the invention may also have dual activity
as agonists of GPR119 and inhibitors of DPP-IV.
SUMMARY OF THE INVENTION
[0013] The present invention is directed to compounds which have
activity as agonists of GPR119 and may also be inhibitors of DPP-IV
and are useful for the treatment of metabolic disorders including
type II diabetes.
DETAILED DESCRIPTION OF THE INVENTION
[0014] The present invention provides compounds of formula (I) and
pharmaceutically acceptable salts thereof:
##STR00001##
[0015] wherein p is 1 or 2;
[0016] when p is 2, Z is N--C(O)OR.sup.4, N--C(O)NR.sup.4R.sup.5 or
N-heteroaryl which may optionally be substituted by one or two
groups selected from C.sub.1-4 alkyl, C.sub.3-6 cycloalkyl
optionally substituted by C.sub.1-4 alkyl, C.sub.1-4 alkoxy,
C.sub.1-4 haloalkyl and halogen;
[0017] when p is 1, Z can also be --N--CH.sub.2-phenyl wherein the
phenyl is optionally substituted by 1 or 2 groups independently
selected from C.sub.1-4 alkyl, C.sub.1-4 haloalkyl and halo;
[0018] A is a para-substituted phenyl or a para-substituted
6-membered heteroaryl ring containing one or two nitrogen
atoms;
[0019] B is a 5-membered heteroaryl ring containing one of more
heteroatoms selected from N, O and S or, a para-substituted
6-membered heteroaryl ring containing one or two nitrogens;
[0020] when B is a 5-membered heteroaryl ring X is --O--CR.sup.6H--
or --CR.sup.7H--O--CR.sup.6H--; and when B is a 6-membered
heteroaryl ring X is --O-- or CR.sup.6H--O--;
[0021] R.sup.1 is hydrogen, halo, cyano, C.sub.1-4 alkyl or
C.sub.1-4 haloalkyl;
[0022] q is 1 or 2;
[0023] R.sup.2 is
##STR00002##
phenyl optionally substituted by one or more halo groups, or
pyridyl optionally substituted by one or more halo or methyl
groups;
[0024] R.sup.3 is independently halo or methyl;
[0025] n is 0 or 1;
[0026] m is 0, 1 or 2;
[0027] R.sup.4 is C.sub.2-6 alkyl or C.sub.3-6 cycloalkyl wherein
the cycloalkyl is optionally substituted by C.sub.1-4alkyl;
[0028] R.sup.5 is hydrogen or C.sub.1-4 alkyl; and
[0029] R.sup.6 and R.sup.7 are independently hydrogen or C.sub.1-2
alkyl.
[0030] In a preferred embodiment the compounds of the invention
have the stereochemistry as defined in formula (Ia), such compounds
demonstrate DPP-IV inhibitory activity:
##STR00003##
[0031] In one of embodiment of the invention each p is
independently 1 or 2, i.e. forming a 4-, 5- or 6-membered ring. In
another embodiment of the invention each p is the same, i.e.
forming a 4- or 6-membered ring. In the compounds of the invention
p is preferably 2.
[0032] In one embodiment of the invention Z is N--C(O)OR.sup.4.
[0033] In a further embodiment of the invention Z is N-heteroaryl
which may optionally be substituted by one or two groups selected
from C.sub.1-4 alkyl, C.sub.3-6 cycloalkyl optionally substituted
by C.sub.1-4alkyl, C.sub.1-4 alkoxy, C.sub.1-4 haloalkyl and
halogen.
[0034] When Z is N-heteroaryl preferred heteroaryl groups include
oxadiazole and pyrimidine.
[0035] A is preferably phenyl, pyridyl or pyrimidinyl.
[0036] R.sup.1 is preferably hydrogen.
[0037] In one embodiment of the invention B is a 5-membered
heteroaryl ring, in another B is a 6-membered heteroaryl ring.
[0038] When B is a 5-membered heteroaryl ring X is preferably
--O--CR.sup.6H--; and when B is a 6-membered heteroaryl ring X is
preferably or CR.sup.6H--O--.
[0039] R.sup.2 is preferably phenyl or pyridyl, more preferably
phenyl, and even more preferably substituted phenyl.
[0040] When R.sup.2 is phenyl substituted by one or more halo
groups it is preferably substituted by 1 to 3 halo groups, the halo
groups are preferably fluoro or chloro, more preferably fluoro.
[0041] When R.sup.2 is pyridyl it is preferably 2-pyridyl.
[0042] When R.sup.2 is substituted pyridyl it is preferably
substituted by 1 to 3 halo or methyl groups, more preferably 1 or 2
methyl groups.
[0043] n is preferably 1.
[0044] R.sup.4 is preferably C.sub.2-6 alkyl.
[0045] R.sup.6 and R.sup.7 are independently preferably hydrogen or
methyl.
[0046] A group of compounds which may be mentioned are those of
formula (Ib) and pharmaceutically acceptable salts thereof:
##STR00004##
[0047] wherein p is 1 or 2;
[0048] when p is 2, Z is N--C(O)OR.sup.4, N--C(O)NR.sup.4R.sup.5 or
N-heteroaryl which may optionally be substituted by one or two
groups selected from C.sub.1-4 alkyl, C.sub.1-4 alkoxy, C.sub.1-4
haloalkyl and halogen;
[0049] when p is 1, Z is --N--CH.sub.2-phenyl wherein the phenyl is
optionally substituted by 1 or 2 groups independently selected from
C.sub.1-4alkyl, C.sub.1-4haloalkyl and halo;
[0050] A is a para-substituted phenyl or a para-substituted
6-membered heteroaryl ring containing one or two nitrogen
atoms;
[0051] B is a 5-membered heteroaryl ring containing one of more
heteroatoms selected from N, O and S or, a para-substituted
6-membered heteroaryl ring containing one or two nitrogens;
[0052] when B is a 5-membered heteroaryl ring X is --O--CR.sup.6H--
and when B is a 6-membered heteroaryl ring X is --O-- or
CR.sup.6H--O--;
[0053] R.sup.1 is hydrogen, halo, cyano, C.sub.1-4alkyl or
C.sub.1-4haloalkyl;
[0054] q is 1 or 2;
[0055] R.sup.2 is
##STR00005##
or phenyl optionally substituted by one or more halo groups;
[0056] R.sup.3 is independently halo or methyl;
[0057] n is 0 or 1;
[0058] m is 0, 1 or 2;
[0059] R.sup.4 is C.sub.2-6 alkyl;
[0060] R.sup.5 is C.sub.1-4alkyl; and
[0061] R.sup.6 is hydrogen or C.sub.1-2alkyl.
[0062] In a preferred embodiment of the compounds formula (Ib) they
have the stereochemistry as defined in formula (Ia).
[0063] While the preferred groups for each variable have generally
been listed above separately for each variable, preferred compounds
of this invention include those in which several or each variable
in formula (I) is selected from the preferred groups for each
variable. Therefore, this invention is intended to include all
combinations of preferred listed groups.
[0064] The molecular weight of the compounds of the invention is
preferably less than 800, more preferably less than 600.
[0065] As used herein, unless stated otherwise, "alkyl" means
carbon chains which may be linear or branched. Examples of alkyl
groups include ethyl, propyl, isopropyl, butyl, sec- and
tert-butyl.
[0066] The term "heteroaryl" rings means 5- or 6-membered
N-containing heteroaryl rings containing up to 2 additional
heteroatoms selected from N, O and S. Examples of such heteroaryl
rings are pyrrolyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl,
thiazolyl, isothiazolyl, triazolyl, oxadiazolyl, thiadiazolyl,
pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl and triazinyl.
[0067] Reference to para substitution in relation to rings A and B
refers to the positions of the group --B-- and the N-containing
heterocycle on ring A and groups --X-- and -A- on ring B.
[0068] Compounds described herein may contain one or more
asymmetric centers and may thus give rise to diastereomers and
optical isomers. The present invention includes all such possible
diastereomers as well as their racemic mixtures, their
substantially pure resolved enantiomers, all possible geometric
isomers, and pharmaceutically acceptable salts thereof. The present
invention includes all stereoisomers of the compounds of the
invention and pharmaceutically acceptable salts thereof. Further,
mixtures of stereoisomers as well as isolated specific
stereoisomers are also included. During the course of the synthetic
procedures used to prepare such compounds, or in using racemization
or epimerization procedures known to those skilled in the art, the
products of such procedures can be a mixture of stereoisomers.
[0069] When a tautomer of the compound of the invention exists, the
present invention includes any possible tautomers and
pharmaceutically acceptable salts thereof, and mixtures thereof,
except where specifically drawn or stated otherwise.
[0070] When the compound of the invention and pharmaceutically
acceptable salts thereof exist in the form of solvates or
polymorphic forms, the present invention includes any possible
solvates and polymorphic forms. A type of a solvent that forms the
solvate is not particularly limited so long as the solvent is
pharmacologically acceptable. For example, water, ethanol,
propanol, acetone or the like can be used.
[0071] The term "pharmaceutically acceptable salts" refers to salts
prepared from pharmaceutically acceptable non-toxic bases or acids.
When the compound of the present invention is acidic, its
corresponding salt can be conveniently prepared from
pharmaceutically acceptable non-toxic bases, including inorganic
bases and organic bases. Salts derived from such inorganic bases
include aluminum, ammonium, calcium, copper (ic and ous), ferric,
ferrous, lithium, magnesium, potassium, sodium, zinc and the like
salts. Particularly preferred are the ammonium, calcium, magnesium,
potassium and sodium salts. Salts derived from pharmaceutically
acceptable organic non-toxic bases include salts of primary,
secondary, and tertiary amines, as well as cyclic amines and
substituted amines such as naturally occurring and synthesized
substituted amines. Other pharmaceutically acceptable organic
non-toxic bases from which salts can be formed include arginine,
betaine, caffeine, choline, N',N'-dibenzylethylenediamine,
diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol,
ethanolamine, ethylenediamine, N-ethylmorpholine,
N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine,
isopropylamine, lysine, methylglucamine, morpholine, piperazine,
piperidine, polyamine resins, procaine, purines, theobromine,
triethylamine, trimethylamine, tripropylamine, tromethamine and the
like.
[0072] When the compound of the invention is basic, its
corresponding salt can be conveniently prepared from
pharmaceutically acceptable non-toxic acids, including inorganic
and organic acids. Such acids include, for example, acetic,
benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic,
fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic,
lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric,
pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric,
p-toluenesulfonic acid and the like
[0073] Since the compounds of the invention are intended for
pharmaceutical use they are preferably provided in substantially
pure form, for example at least 60% pure, more suitably at least
75% pure, especially at least 98% pure (% are on a weight for
weight basis).
[0074] The compounds of formula (I) can be prepared as described
below, wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5,
R.sup.6, A, B, X, Z, m, n, p, q are as defined for formula (I). PG
is a protecting group, Hal is halogen and E is either halogen or
triflate.
[0075] Compounds of formula (I) can be prepared as outlined in
Scheme 1. Compounds of formula (IV) can be prepared by SN.sub.Ar
displacement of suitable haloaromatic compounds of formula (II)
with amines of formula (III) under standard conditions, for
example, DBU and DMSO at 120.degree. C. Alternatively, compounds of
formula (IV) can be prepared by reaction of suitable haloaromatic
compounds of formula (II) with amines of formula (III) under
Buchwald-Hartwig conditions, such as, Pd.sub.2(dba).sub.3 and BINAP
in a suitable solvent, such as toluene at 110.degree. C.
Deprotection of the amine functionality, using standard conditions
well known to those with skill in the art, affords compounds of
formula (I) as described above.
##STR00006##
[0076] Building blocks of formula (II), where B is a
para-substituted 6-membered heteroaryl ring containing one or two
nitrogens and X is --O-- or CR.sup.6H--O--, can be prepared as
outlined in Scheme 2. Aryl halides of formula (V) can be treated
with boronates of formula (VI) under standard Suzuki conditions,
for example, [1,1-bis(diphenylphosphino)ferrocene]dichloropalladium
in a suitable solvent such as DMF/water at 80.degree. C.
##STR00007##
[0077] Alternatively, building blocks of formula (II), where B is a
para-substituted 6-membered heteroaryl ring containing one or two
nitrogens and X is --O-- or CR.sup.6H--O--, can be prepared as
outlined in Scheme 3. Aryl boronates of formula (VII) can be
prepared by reaction of aryl halide of formula (V) and
bis(pinacolato)diboron in the presence of a suitable catalyst, such
as [1,1-bis(diphenylphosphino)ferrocene]dichloropalladium in a
suitable solvent such as 1,4-dioxane at 110.degree. C. Building
blocks of formula (II) can be prepared by reaction of boronates of
formula (VII) with aryl halides or aryl triflates of formula (VIII)
under standard Suzuki conditions, for example,
[1,1-bis(diphenylphosphino)ferrocene]dichloropalladium in a
suitable solvent such as DMF/water at 80.degree. C.
##STR00008##
[0078] Building blocks of formula (V) where B is a para-substituted
6-membered heteroaryl ring containing one or two nitrogens and X is
--O-- or CR.sup.6H--O--, can be prepared as outlined in Scheme 4.
Alcohols of formula (IX) can be treated with hydroxyaryls of
formula (X) under standard Mitsonobu conditions, for example, using
azodicarboxylic dipiperidide and tributylphosphine in a suitable
solvent such as toluene.
##STR00009##
[0079] Alternatively, building blocks of formula (V) where B is a
para-substituted 6-membered heteroaryl ring containing one or two
nitrogens and X is --O-- or CR.sup.6H--O--, can be prepared as
outlined in Scheme 5. Alcohols of formula (IX) can be treated with
a suitable dihaloaryl compound of formula (XI) under standard
SN.sub.Ar conditions, such as DBU and DMSO at 120.degree. C.
##STR00010##
[0080] Building blocks of formula (II) where B is a
1,2,4-oxadiazol-5-yl and X is --O--CR.sup.6H-- can be prepared as
outlined in Scheme 6. Amidoxime of formula (XII) can be prepared by
reaction of nitrile of formula (XIII) and hydroxylamine
hydrochloride in the presence of a suitable base such as
K.sub.2CO.sub.3 in a suitable solvent such as ethanol/water at
78.degree. C. Building blocks of formula (II) as described above
can be prepared by reaction of amidoxime of formula (XII) with acid
of formula (XIV) under standard conditions, such as isobutyl
chloroformate and triethylamine, in a suitable solvent such as
DMF.
##STR00011##
[0081] Building blocks of formula (II) where B is a
1,2,4-oxadiazol-3-yl and X is --O--CR.sup.6H-- can be prepared as
outlined in Scheme 7. Amidoximes of formula (XV) can be prepared by
reaction of nitrile of formula (XVI) and hydroxylamine
hydrochloride in the presence of a suitable base such as
K.sub.2CO.sub.3 in a suitable solvent such as ethanol/water at
78.degree. C. Building blocks of formula (II) as described above
can be prepared by reaction of amidoxime of formula (XV) with acid
of formula (XVII) under standard conditions, such as isobutyl
chloroformate and triethylamine, in a suitable solvent such as
DMF.
##STR00012##
[0082] Building blocks of formula (II) where B is a thiazol-2-yl
and X is --O--CR.sup.6H-- can be prepared as outlined in Scheme 8.
Primary amides of formula (XVIII) can be prepared by reaction of
acids of formula (XIV) with ammonia in 1,4-dioxane solution under
standard amide coupling conditions, for example, HOBT and EDCI, in
a suitable solvent, such as DCM. Thioamides of formula (XIX) can be
prepared by reaction of primary amides of formula (XVIII) under
standard conditions, for example using Lawesson's reagent in a
suitable solvent such as toluene at reflux. Building blocks of
formula (II) as described above can be prepared by reaction of
bromoketones of formula (XX) with thioamide of formula (XIX) under
standard Hantzsch conditions, for example ethanol at room
temperature.
##STR00013##
[0083] Building blocks of formula (II) where B is a thiazol-5-yl
and X is --O--CR.sup.6H-- can be prepared as outlined in Scheme 9.
Primary amides of formula (XXI) can be prepared by reaction of
acids of formula (XVII) with ammonia in 1,4-dioxane solution under
standard amide coupling conditions, for example, HOBT and EDCI, in
a suitable solvent, such as DCM. Thioamides of formula (XXII) can
be prepared by reaction of primary amides of formula (XVIII) under
standard conditions, for example using Lawesson's reagent in a
suitable solvent such as toluene at reflux. Building blocks of
formula (II) as described above can be prepared by reaction of
chloroketones of formula (XXIII) with thioamide of formula (XXII)
under standard Hantzsch conditions, for example ethanol at room
temperature.
##STR00014##
[0084] Building blocks of formula (XVI) where X is --O--CR.sup.6H--
can be prepared as outlined in Scheme 10. Alcohols of formula
(XXIV) can be treated with bromides of formula (XXV) under standard
conditions, for example, NaH in a suitable solvent, such as THF at
0.degree. C.
##STR00015##
[0085] Building blocks of formula (XX) can be prepared as outlined
in Scheme 11. Ketones of formula (XXVI) can be treated with
trimethylphenylammonium tribromide in a suitable solvent, such as
THF.
##STR00016##
[0086] Building blocks of formula (XXIII) where X is
--O--CR.sup.6H-- can be prepared as outlined in Scheme 12. Alcohols
of formula (XXIV) can be treated with 1,3-dichloroacetone in the
presence of a suitable base, such as K.sub.2CO.sub.3, in a suitable
solvent such as DMF.
##STR00017##
[0087] Examples and syntheses of building blocks of formula (III)
have been described elsewhere: Benbow et.al., WO2007/148185;
Brackes et.al., Bioorg. Med. Chem. Lett., 2007, 17 2005-2012; Pei
et.al., J. Med. Chem., 2007, 50 (8), 1983-1987; Cox et.al., Bioorg.
Med. Chem. Lett., 2007, 17 4579-4583; Wright et.al., Bioorg. Med.
Chem. Lett., 2007, 17 5638-5642.
[0088] The synthesis of building blocks of formula (IX) where p is
2 and X is --O-- or CR.sup.6H--O-- have been described elsewhere:
Fang et. al., WO2008/070692; Alper et. al., WO2008/097428; Wacker
et.al., WO2009/012275.
[0089] The synthesis of building blocks of formula (IX) where p is
1 and X is --O-- or CR.sup.6H--O-- have been described elsewhere:
Arnould et.al., WO2007/091046; Evans et.al., WO2008/079028.
[0090] The synthesis of building blocks of formula (XIV) where X is
--O--CR.sup.6H-- have been described elsewhere: Bertram et.al.,
WO2007/116229.
[0091] Other compounds of formula (I) may be prepared by methods
analogous to those described above or by methods known per se.
Further details for the preparation of the compounds of formula (I)
are found in the examples.
[0092] The compounds of formula (I) may be prepared singly or as
compound libraries comprising at least 2, for example 5 to 1,000,
compounds and more preferably 10 to 100 compounds of formula (I).
Compound libraries may be prepared by a combinatorial "split and
mix" approach or by multiple parallel syntheses using either
solution or solid phase chemistry, using procedures known to those
skilled in the art.
[0093] During the synthesis of the compounds of formula (I), labile
functional groups in the intermediate compounds, e.g. hydroxy,
carboxy and amino groups, may be protected. The protecting groups
may be removed at any stage in the synthesis of the compounds of
formula (I) or may be present on the final compound of formula (I).
A comprehensive discussion of the ways in which various labile
functional groups may be protected and methods for cleaving the
resulting protected derivatives is given in, for example,
Protective Groups in Organic Chemistry, T. W. Greene and P. G. M.
Wuts, (1991) Wiley-Interscience, New York, 2.sup.nd edition.
[0094] The processes for the production of the compounds of formula
(I) and intermediates thereto as described above are also included
as further aspects of the present invention.
[0095] Any novel intermediates as defined in the Schemes above or
in the Examples, are also included within the scope of the
invention. Therefore according to a further aspect of the invention
there is provided a compound of any one of formulae (II), (IV),
(V), (XIV), (XV), (XVI), (XVIII), (XIX) and (XXIII) as defined
above. The preferred groups for variables recited above in relation
to the compounds of formula (I) also apply to the intermediates
compounds.
[0096] As indicated above the compounds of the invention are useful
as GPR119 agonists, e.g. for the treatment and/or prophylaxis of
diabetes. For such use the compounds of the invention will
generally be administered in the form of a pharmaceutical
composition.
[0097] The compounds of the invention may also be useful as dual
GPR119 agonists/DPP-IV inhibitors, e.g. for the treatment and/or
prophylaxis of diabetes. For such use the compounds of the
invention will generally be administered in the form of a
pharmaceutical composition.
[0098] The invention also provides a compound of the invention, or
a pharmaceutically acceptable salt thereof, for use as a
pharmaceutical.
[0099] The invention also provides a pharmaceutical composition
comprising a compound of the invention, in combination with a
pharmaceutically acceptable carrier.
[0100] Preferably the composition is comprised of a
pharmaceutically acceptable carrier and a non-toxic therapeutically
effective amount of a compound of the invention, or a
pharmaceutically acceptable salt thereof.
[0101] Moreover, the invention also provides a pharmaceutical
composition for the treatment of disease by modulating GPR119 and
optionally DPP-IV, resulting in the prophylactic or therapeutic
treatment of diabetes, comprising a pharmaceutically acceptable
carrier and a non-toxic therapeutically effective amount of
compound of the invention, or a pharmaceutically acceptable salt
thereof.
[0102] The pharmaceutical compositions may optionally comprise
other therapeutic ingredients or adjuvants. The compositions
include compositions suitable for oral, rectal, topical, and
parenteral (including subcutaneous, intramuscular, and intravenous)
administration, although the most suitable route in any given case
will depend on the particular host, and nature and severity of the
conditions for which the active ingredient is being administered.
The pharmaceutical compositions may be conveniently presented in
unit dosage form and prepared by any of the methods well known in
the art of pharmacy.
[0103] In practice, the compounds of the invention, or
pharmaceutically acceptable salts thereof, can be combined as the
active ingredient in intimate admixture with a pharmaceutical
carrier according to conventional pharmaceutical compounding
techniques. The carrier may take a wide variety of forms depending
on the form of preparation desired for administration, e.g. oral or
parenteral (including intravenous).
[0104] Thus, the pharmaceutical compositions can be presented as
discrete units suitable for oral administration such as capsules,
cachets or tablets each containing a predetermined amount of the
active ingredient. Further, the compositions can be presented as a
powder, as granules, as a solution, as a suspension in an aqueous
liquid, as a non-aqueous liquid, as an oil-in-water emulsion, or as
a water-in-oil liquid emulsion. In addition to the common dosage
forms set out above, the compound of the invention, or a
pharmaceutically acceptable salt thereof, may also be administered
by controlled release means and/or delivery devices. The
compositions may be prepared by any of the methods of pharmacy. In
general, such methods include a step of bringing into association
the active ingredient with the carrier that constitutes one or more
necessary ingredients. In general, the compositions are prepared by
uniformly and intimately admixing the active ingredient with liquid
carriers or finely divided solid carriers or both. The product can
then be conveniently shaped into the desired presentation.
[0105] The compounds of the invention, or pharmaceutically
acceptable salts thereof, can also be included in pharmaceutical
compositions in combination with one or more other therapeutically
active compounds.
[0106] The pharmaceutical carrier employed can be, for example, a
solid, liquid, or gas. Examples of solid carriers include lactose,
terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium
stearate, and stearic acid. Examples of liquid carriers are sugar
syrup, peanut oil, olive oil, and water. Examples of gaseous
carriers include carbon dioxide and nitrogen.
[0107] In preparing the compositions for oral dosage form, any
convenient pharmaceutical media may be employed. For example,
water, glycols, oils, alcohols, flavoring agents, preservatives,
coloring agents, and the like may be used to form oral liquid
preparations such as suspensions, elixirs and solutions; while
carriers such as starches, sugars, microcrystalline cellulose,
diluents, granulating agents, lubricants, binders, disintegrating
agents, and the like may be used to form oral solid preparations
such as powders, capsules and tablets. Because of their ease of
administration, tablets and capsules are the preferred oral dosage
units whereby solid pharmaceutical carriers are employed.
Optionally, tablets may be coated by standard aqueous or nonaqueous
techniques.
[0108] A tablet containing the composition of this invention may be
prepared by compression or molding, optionally with one or more
accessory ingredients or adjuvants. Compressed tablets may be
prepared by compressing, in a suitable machine, the active
ingredient in a free-flowing form such as powder or granules,
optionally mixed with a binder, lubricant, inert diluent, surface
active or dispersing agent. Molded tablets may be made by molding
in a suitable machine, a mixture of the powdered compound moistened
with an inert liquid diluent. Each tablet preferably contains from
about 0.05 mg to about 5 g of the active ingredient and each cachet
or capsule preferably containing from about 0.05 mg to about 5 g of
the active ingredient.
[0109] For example, a formulation intended for the oral
administration to humans may contain from about 0.5 mg to about 5 g
of active agent, compounded with an appropriate and convenient
amount of carrier material which may vary from about 5 to about 95
percent of the total composition. Unit dosage forms will generally
contain between from about 1 mg to about 2 g of the active
ingredient, typically 25 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg,
500 mg, 600 mg, 800 mg, or 1000 mg.
[0110] Pharmaceutical compositions of the present invention
suitable for parenteral administration may be prepared as solutions
or suspensions of the active compounds in water. A suitable
surfactant can be included such as, for example,
hydroxypropylcellulose. Dispersions can also be prepared in
glycerol, liquid polyethylene glycols, and mixtures thereof in
oils. Further, a preservative can be included to prevent the
detrimental growth of microorganisms.
[0111] Pharmaceutical compositions of the present invention
suitable for injectable use include sterile aqueous solutions or
dispersions. Furthermore, the compositions can be in the form of
sterile powders for the extemporaneous preparation of such sterile
injectable solutions or dispersions. In all cases, the final
injectable form must be sterile and must be effectively fluid for
easy syringability. The pharmaceutical compositions must be stable
under the conditions of manufacture and storage; thus, preferably
should be preserved against the contaminating action of
microorganisms such as bacteria and fungi. The carrier can be a
solvent or dispersion medium containing, for example, water,
ethanol, polyol (e.g. glycerol, propylene glycol and liquid
polyethylene glycol), vegetable oils, and suitable mixtures
thereof.
[0112] Pharmaceutical compositions of the present invention can be
in a form suitable for topical use such as, for example, an
aerosol, cream, ointment, lotion, dusting powder, or the like.
Further, the compositions can be in a form suitable for use in
transdermal devices. These formulations may be prepared, using a
compound of the invention, or a pharmaceutically acceptable salt
thereof, via conventional processing methods. As an example, a
cream or ointment is prepared by admixing hydrophilic material and
water, together with about 5 wt % to about 10 wt % of the compound,
to produce a cream or ointment having a desired consistency.
[0113] Pharmaceutical compositions of this invention can be in a
form suitable for rectal administration wherein the carrier is a
solid. It is preferable that the mixture forms unit dose
suppositories. Suitable carriers include cocoa butter and other
materials commonly used in the art. The suppositories may be
conveniently formed by first admixing the composition with the
softened or melted carrier(s) followed by chilling and shaping in
molds.
[0114] In addition to the aforementioned carrier ingredients, the
pharmaceutical formulations described above may include, as
appropriate, one or more additional carrier ingredients such as
diluents, buffers, flavoring agents, binders, surface-active
agents, thickeners, lubricants, preservatives (including
anti-oxidants) and the like. Furthermore, other adjuvants can be
included to render the formulation isotonic with the blood of the
intended recipient. Compositions containing a compound of the
invention, or pharmaceutically acceptable salts thereof, may also
be prepared in powder or liquid concentrate form.
[0115] Generally, dosage levels on the order of 0.01 mg/kg to about
150 mg/kg of body weight per day are useful in the treatment of the
above-indicated conditions, or alternatively about 0.5 mg to about
7 g per patient per day. For example, obesity may be effectively
treated by the administration of from about 0.01 to 50 mg of the
compound per kilogram of body weight per day, or alternatively
about 0.5 mg to about 3.5 g per patient per day.
[0116] It is understood, however, that the specific dose level for
any particular patient will depend upon a variety of factors
including the age, body weight, general health, sex, diet, time of
administration, route of administration, rate of excretion, drug
combination and the severity of the particular disease undergoing
therapy.
[0117] The compounds of the invention may be used in the treatment
of diseases or conditions in which GPR119 and optionally DPP-IV
play a role.
[0118] Thus the invention also provides a method for the treatment
of a disease or condition in which GPR119 and optionally DPP-IV
play a role comprising a step of administering to a subject in need
thereof an effective amount of a compound of the invention, or a
pharmaceutically acceptable salt thereof. Such diseases or
conditions diabetes, obesity, impaired glucose tolerance, insulin
resistance and diabetic complications such as neuropathy,
nephropathy, retinopathy, cataracts, cardiovascular complications
and dyslipidaemia). And the treatment of patients who have an
abnormal sensitivity to ingested fats leading to functional
dyspepsia. The compounds of the invention may also be used for
treating metabolic diseases such as metabolic syndrome (syndrome
X), impaired glucose tolerance, hyperlipidemia,
hypertriglyceridemia, hypercholesterolemia, low HDL levels and
hypertension.
[0119] The invention also provides a method for the treatment of
type II diabetes, comprising a step of administering to a patient
in need thereof an effective amount of a compound of the invention,
or a pharmaceutically acceptable salt thereof.
[0120] The invention also provides a method for the treatment of
obesity, metabolic syndrome (syndrome X), impaired glucose
tolerance, hyperlipidemia, hypertriglyceridemia,
hypercholesterolemia, low HDL levels or hypertension comprising a
step of administering to a patient in need thereof an effective
amount of a compound of the invention, or a pharmaceutically
acceptable salt thereof.
[0121] The invention also provides a compound of the invention, or
a pharmaceutically acceptable salt thereof, for use in the
treatment of a condition as defined above.
[0122] The invention also provides the use of a compound of the
invention, or a pharmaceutically acceptable salt thereof, in the
manufacture of a medicament for the treatment of a condition as
defined above.
[0123] In the methods of the invention the term "treatment"
includes both therapeutic and prophylactic treatment.
[0124] The compounds of the invention may exhibit advantageous
properties compared to known compounds or combination therapies for
the treatment of diabetes.
[0125] The compounds of the invention, or pharmaceutically
acceptable salts thereof, may be administered alone or in
combination with one or more other therapeutically active
compounds. The other therapeutically active compounds may be for
the treatment of the same disease or condition as the compounds of
the invention or a different disease or condition. The
therapeutically active compounds may be administered
simultaneously, sequentially or separately.
[0126] The compounds of the invention may be administered with
other active compounds for the treatment of obesity and/or
diabetes, for example insulin and insulin analogs, gastric lipase
inhibitors, pancreatic lipase inhibitors, sulfonyl ureas and
analogs, biguanides e.g. metformin, .alpha.2 agonists, glitazones,
PPAR-.gamma. agonists, mixed PPAR-.alpha./.gamma. agonists, RXR
agonists, fatty acid oxidation inhibitors, .alpha.-glucosidase
inhibitors, .beta.-agonists, phosphodiesterase inhibitors, lipid
lowering agents, glycogen phosphorylase inhibitors, antiobesity
agents e.g. pancreatic lipase inhibitors, MCH-1 antagonists and
CB-1 antagonists (or inverse agonists), amylin antagonists,
lipoxygenase inhibitors, somostatin analogs, glucokinase
activators, glucagon antagonists, insulin signalling agonists,
PTP1B inhibitors, gluconeogenesis inhibitors, antilypolitic agents,
GSK inhibitors, galanin receptor agonists, anorectic agents, CCK
receptor agonists, leptin, serotonergic/dopaminergic antiobesity
drugs, reuptake inhibitors e.g. sibutramine, CRF antagonists, CRF
binding proteins, thyromimetic compounds, aldose reductase
inhibitors, glucocorticoid receptor antagonists, NHE-1 inhibitors
or sorbitol dehydrogenase inhibitors.
[0127] Combination therapy comprising the administration of a
compound of the invention, or a pharmaceutically acceptable salt
thereof, and at least one other agent, for example another agent
for the treatment of diabetes or obesity, represents a further
aspect of the invention.
[0128] The present invention also provides a method for the
treatment of diabetes in a mammal, such as a human, which method
comprises administering an effective amount of a compound of the
invention, or a pharmaceutically acceptable salt thereof, and
another agent, for example another agent for the treatment of
diabetes or obesity, to a mammal in need thereof.
[0129] The invention also provides the use of a compound of the
invention, or a pharmaceutically acceptable salt thereof, and
another agent for the treatment of diabetes.
[0130] The invention also provides the use of a compound of the
invention, or a pharmaceutically acceptable salt thereof, in the
manufacture of a medicament for use in combination with another
agent, for the treatment of diabetes.
[0131] The compound of the invention, or a pharmaceutically
acceptable salt thereof, and the other agent(s) may be
co-administered or administered sequentially or separately.
[0132] Co-administration includes administration of a formulation
which includes both the compound of the invention, or a
pharmaceutically acceptable salt thereof, and the other agent(s),
or the simultaneous or separate administration of different
formulations of each agent. Where the pharmacological profiles of
the compound of the invention, or a pharmaceutically acceptable
salt thereof, and the other agent(s) allow it, coadministration of
the two agents may be preferred.
[0133] The invention also provides the use of a compound of the
invention, or a pharmaceutically acceptable salt thereof, and
another agent in the manufacture of a medicament for the treatment
of diabetes.
[0134] The invention also provides a pharmaceutical composition
comprising a compound of the invention, or a pharmaceutically
acceptable salt thereof, and another antidiabetic agent, and a
pharmaceutically acceptable carrier. The invention also encompasses
the use of such compositions in the methods described above.
[0135] All publications, including, but not limited to, patents and
patent application cited in this specification, are herein
incorporated by reference as if each individual publication were
specifically and individually indicated to be incorporated by
reference herein as fully set forth.
[0136] The invention will now be described by reference to the
following examples which are for illustrative purposes and are not
to be construed as a limitation of the scope of the present
invention.
EXAMPLES
Materials and Methods
[0137] Column chromatography was carried out on SiO.sub.2 (40-63
mesh) unless specified otherwise. LCMS data were obtained as
follows: Atlantis 3.mu. C.sub.18 column (3.0.times.20.0 mm, flow
rate=0.85 mL/min) eluting with a H.sub.2O-MeCN solution containing
0.1% HCO.sub.2H over 6 min with UV detection at 220 nm. Gradient
information: 0.0-0.3 min 100% H.sub.2O; 0.3-4.25 min: Ramp up to
10% H.sub.2O-90% MeCN; 4.25-4.4 min: Ramp up to 100% MeCN; 4.4-4.9
min: Hold at 100% MeCN; 4.9-6.0 min: Return to 100% H.sub.2O. The
mass spectra were obtained using an electrospray ionisation source
in either the positive (ES.sup.+) or negative (ES.sup.-) ion
modes.
[0138] LCMS-method 2 data were obtained as follows: Xbridge C18
column (2.1.times.50 mm, 2.5 .mu.M, flow rate 0.8 mL/min) eluting
with an MeCN-10 mM NH.sub.4HCO.sub.3 solution over 1.5 min with UV
detection at 215-350 nm. Gradient information: 0-0.8 min: 98% MeCN
2% NH.sub.4HCO.sub.3 to 98% NH.sub.4HCO.sub.3 2% MeCN; 0.8-1.2 min:
hold at 98% NH.sub.4HCO.sub.3 2% MeCN. The mass spectra were
obtained using an electrospray ionisation source in the positive
(ES.sup.+) mode.
[0139] LCMS-method 3 data were obtained as follows: Xbridge C18
column (2.1.times.5.0 mm, 2.55 .mu.M, flow rate 0.8 mL/min) eluting
with an MeCN-10 mM NH.sub.4HCO.sub.3 solution over 5 min with UV
detection at 215-350 nm. Gradient information: 0-4 min: 98% MeCN 2%
NH.sub.4HCO.sub.3 to 98% NH.sub.4HCO.sub.3 2% MeCN; 4-4.6 min: hold
at 98% NH.sub.4HCO.sub.3 2% MeCN. The mass spectra were obtained
using an electrospray ionisation source in the positive (ES.sup.+)
mode.
[0140] LCMS-method 4 data were obtained as follows: Xbridge C18
column (3.0.times.150 mm, 5 .mu.M, flow rate 1.0 mL/min) eluting
with an MeCN-10 mM NH.sub.4HCO.sub.3 solution over 5 min with UV
detection at 215-350 nm. Gradient information: 0-0.1 min: hold at
5% MeCN 95% NH.sub.4HCO.sub.3; 0.1-3.0 min: 5% MeCN 95%
NH.sub.4HCO.sub.3 to 5% NH.sub.4HCO.sub.3 95% MeCN; 3.0-3.9 min:
hold at 5% NH.sub.4HCO.sub.3 95% MeCN. The mass spectra were
obtained using an electrospray ionisation source in the positive
(ES.sup.+) mode.
[0141] Chiral-HPLC was performed on a Daicel chiralpak IA
250.times.20 mm, 5 .mu.M column.
[0142] Abbreviations and acronyms: AcOH: Acetic acid; atm:
Atmospheres; BA: n-butylamine; CHCl.sub.3: Chloroform; DBU:
1,8-Diazabicyclo[5.4.0]undec-7-ene; DCM: Dichloromethane; DEA:
Diethylamine; DIPEA: Diisopropylethylamine; DMAP:
Dimethylpyridin-4-ylamine; DMF: Dimethylformamide; DMSO:
Dimethylsulfoxide; EDCI: (3-Dimethylaminopropyl)ethylcarbodiimide
hydrochloride; EtOAc: Ethyl Acetate; Et.sub.2O: Diethyl ether;
EtOH: Ethanol; h: hour(s); HO: Hydrochloric acid; HCO.sub.2H:
Formic acid; H.sub.2O: Water; HOBt: 1-Hydroxybenzotriazole
monohydrate; HPLC: High performance liquid chromatography; IH:
Isohexane; IMS: Industrial methylated spirit; IPA: Isopropyl
alcohol; M: Molar; MeCN: Acetonitrile; MeOH: Methanol; MgSO.sub.4:
Magnesium sulphate; min: minute/s; MTBE: Methyl-tert-butyl ether;
NaHCO.sub.3: Sodium hydrogen carbonate; Na.sub.2CO.sub.3: Sodium
carbonate; NaOH: Sodium hydroxide; Na.sub.2SO.sub.4: Sodium
sulphate; NH.sub.3: Ammonia; NH.sub.4HCO.sub.3: Ammonium
bicarbonate; NH.sub.4OH: Ammonium hydroxide; Pd: Palladium; RT:
Retention time; r.t.: Room temperature; sat: saturated; SCX: Strong
Cation Exchange resin; SiO.sub.2: Silica gel; THF: Tetrahydrofuran;
TFA: Trifluoroacetic acid; TFAA: Trifluoroacetic anhydride; TsOH:
p-Toluenesulfonic acid monohydrate
[0143] The syntheses of the following compounds have been described
elsewhere: 4-(N-hydroxycarbamimidoylmethoxy)piperidine-1-carboxylic
acid tert-butyl ester: Bradley et. al., WO2007/003961;
4-((R)-1-Carboxyethoxy)piperidine-1-carboxylic acid tert-butyl
ester: Bertram et. al, WO2007/116229;
(3S,4S)-3,4-Diazido-1-benzylpyrrolidine: Benbow et. al.,
WO2007/148185. All other compounds were available from commercial
sources.
Preparation 1
4-[5-(6-Chloropyridin-3-yl)-[1,2,4]oxadiazol-3-ylmethoxy]piperidine-1-carb-
oxylic acid tert-butyl ester
##STR00018##
[0145] To a solution of 6-chloronicotinic acid (500 mg, 3.17 mmol)
in THF (25 mL) was added EDCI (0.74 g, 3.89 mmol), followed by HOBt
(583 mg, 3.81 mmol), and the reaction was stirred at r.t. for 10
min. 4-(N-Hydroxycarbamimidoylmethoxy)piperidine-1-carboxylic acid
tert-butyl ester (866 mg, 3.17 mmol) was added and the reaction was
stirred at r.t. for 16 h before removing the solvent in vacuo. The
resulting residue was partitioned between EtOAc (100 mL) and water
(50 mL). The organic phase was separated, washed with sat.
NaHCO.sub.3 solution, then brine, and dried (MgSO.sub.4), before
removal of the solvent in vacuo. The residue was dissolved in
toluene and the reaction heated to 110.degree. C. for 16 h. Removal
of the solvent in vacuo and purification by column chromatography
(IH:EtOAc, 70:30) afforded the title compound: RT=3.97 min, m/z
(ES.sup.+)=395.2 [M+H].sup.+.
Preparation 2
4-[5-(2-Chloropyrimidin-5-yl)-[1,2,4]oxadiazol-3-ylmethoxy]piperidine-1-ca-
rboxylic acid tert-butyl ester
##STR00019##
[0147] To a solution of 2-chloropyrimidine-5-carboxylic acid (100
mg, 0.63 mmol) in THF (10 mL) was added 1,3-diisopropylcarbodiimide
(99 .mu.L, 0.63 mmol) and the reaction was stirred at r.t. for 10
min. 4-(N-Hydroxycarbamimidoylmethoxy)piperidine-1-carboxylic acid
tert-butyl ester (172 mg, 0.63 mmol) was added and the mixture was
stirred at r.t. for 72 h. The reaction solvent was removed in vacuo
and the resulting residue was re-dissolved in EtOAc (100 mL). The
organic mixture was washed with water, then brine, and dried
(MgSO.sub.4), before removal of the solvent in vacuo. The resulting
residue was dissolved in toluene and heated to 80.degree. C. for 16
h. Removal of the solvent in vacuo and purification by column
chromatography (IH:EtOAc, 60:40) afforded the title compound:
RT=3.77 min, m/z (ES.sup.+)=396.1 [M+H].sup.+.
Preparation 3
4-[3-(6-Chloropyridin-3-yl)-[1,2,4]oxadiazol-5-ylmethoxy]piperidine-1-carb-
oxylic acid tert-butyl ester
##STR00020##
[0149] To a solution of 4-carboxymethoxypiperidine-1-carboxylic
acid tert-butyl ester (1.51 g, 5.83 mmol) in THF (40 mL) was added
EDCI (1.11 g, 6.99 mmol), followed by HOBt (0.95 g, 6.99 mmol), and
the reaction was stirred at r.t. for 10 min.
6-Chloro-N-hydroxynicotinamidine (1.00 g, 5.83 mmol) was added and
the reaction was stirred at r.t. for 17 h before removing the
solvent in vacuo. The resulting residue was partitioned between
EtOAc (200 mL) and water (100 mL), then the organic phase was
separated, washed with sat. NaHCO.sub.3 solution (100 mL), and
dried (MgSO.sub.4) before removal of the solvent in vacuo. The
residue was dissolved in toluene and heated to reflux for 20 h
before removing the solvent in vacuo and re-dissolving the product
in EtOAc (200 mL). The solution was washed with water (100 mL),
then brine (50 mL), and dried (MgSO.sub.4). Removal of the solvent
in vacuo and purification by recrystallisation from EtOAc/IH
afforded the title compound: RT=4.02 min, m/z (ES.sup.+)=395.1
[M+H].sup.+.
Preparation 4
4-{(R)-1-[3-(6-Chloropyridin-3-yl)-[1,2,4]oxadiazol-5-yl]ethoxy}piperidine-
-1-carboxylic acid tert-butyl ester
##STR00021##
[0151] The title compound was prepared by reacting
4-((R)-1-carboxyethoxy)piperidine-1-carboxylic acid tert-butyl
ester with 6-chloro-N-hydroxynicotinamidine employing the procedure
outlined in Preparation 3: RT=4.20 min, m/z (ES.sup.+)=409.2
[M+H].sup.+.
Preparation 5
4-[5-(6-Chloropyridin-3-yl)-[1,2,4]oxadiazol-3-ylmethoxy]piperidine-1-carb-
oxylic acid isopropyl ester
##STR00022##
[0153]
4-[5-(6-Chloropyridin-3-yl)-[1,2,4]oxadiazol-3-ylmethoxy]piperidine-
-1-carboxylic acid tert-butyl ester (Preparation 1, 578 mg, 1.47
mmol) in a solution of HCl in dioxane (4M, 10 mL) was stirred for 2
h before removal of the solvent in vacuo. To a solution of the
product in DCM (20 mL) was added triethylamine (0.66 mL, 4.70 mmol)
and the mixture was cooled to 0.degree. C. A solution of isopropyl
chloroformate in toluene (1M, 1.76 mL, 1.76 mmol) was added,
dropwise, then the resulting reaction was stirred at r.t. for 16 h.
The crude mixture was diluted with DCM (100 mL), washed with water
(50 mL), sat. Na.sub.2CO.sub.3 solution (50 mL), and brine, then
dried (MgSO.sub.4). Removal of the solvent in vacuo afforded the
title compound: RT=3.75 min, m/z (ES.sup.+)=381.2 [M+H].sup.+.
[0154] The following compounds were prepared from the appropriate
tert-butyl carbamate protected compound employing the procedure
outlined in Preparation 5:
TABLE-US-00001 Prep No. Structure Name LCMS 6 ##STR00023## 4-[5-(6-
Chloropyrimidin-5- yl)-[1,2,4]oxadiazol- 3-ylmethoxy]-
piperidine-1- carboxylic acid isopropyl ester RT = 3.54 min, m/z
(ES.sup.+) = 382.1 [M + H].sup.+ 7 ##STR00024## 4-[3-(6-Chloro-
pyridin-3-yl)- [1,2,4]oxadiazol-5- ylmethoxy]piperidine-
1-carboxylic acid isopropyl ester RT = 3.82 min, m/z (ES.sup.+) =
381.1 [M + H].sup.+ 8 ##STR00025## 4-{(R-1-[3-(6-
Chloropyridin-3-yl)- [1,2,4]oxadiazol-5- yl]ethoxy}piperidine-
1-carboxylic acid isopropyl ester RT = 3.98 min, m/z (ES.sup.+) =
395.1 [M + H].sup.+
Preparation 9
4-((R)-1-Carboxyethoxy)piperidine-1-carboxylic acid isopropyl
ester
##STR00026##
[0156] A solution of 4-hydroxypiperidine-1-carboxylic acid
isopropyl ester (10.0 g. 53.4 mmol) in THF (100 mL), in an oven
dried flask, under argon, was cooled to 0.degree. C. Sodium hydride
(60% in mineral oil, 8.55 g, 213.6 mmol) was added, portion-wise,
and the resulting mixture was stirred at 0.degree. C. for 1 h
before stirring at r.t. for 30 min. To the reaction was added a
solution of (S)-2-bromopropionic acid (4.82 g, 53.4 mmol) in THF
(40 mL), dropwise, over 30 min, followed by more THF (60 mL), and
the mixture was stirred at r.t. for 16 h. The reaction was quenched
by the cautious addition of water, then the THF was removed in
vacuo. The resulting aqueous mixture was washed with Et.sub.2O and
acidified to pH1 with 2M HCl. The mixture was extracted with EtOAc
(2.times.150 mL) then the organic fractions were combined, dried
(MgSO.sub.4) and the solvent removed in vacuo to afford the title
compound: .sup.1H NMR .delta..sub.H (400 MHz, CDCl.sub.3):
4.97-4.86 (m, 1H), 4.19-4.09 (m, 1H), 3.89-3.79 (m, 2H), 3.66-3.58
(m, 1H), 3.18-3.08 (m, 2H), 1.91-1.80 (m, 2H), 1.65-1.50 (m, 2H),
1.47 (d, J=7.0 Hz, 3H), 1.26-1.22 (m, 6H).
Preparation 10
4-((R)-1-Carbamoylethoxy)piperidine-1-carboxylic acid isopropyl
ester
##STR00027##
[0158] To a solution of
4-((R)-1-carboxyethoxy)piperidine-1-carboxylic acid isopropyl ester
(Preparation 9, 200 mg, 0.77 mmol) in THF (10 mL), in an oven-dried
flask, under argon, was added EDCI (177 mg, 0.93 mmol), followed by
HOBt (126 mg, 0.93 mmol), and the reaction was stirred at r.t. for
10 min. A solution of NH.sub.3 in dioxane (0.5M, 15 .mu.L, 7.72
mmol) was added and the reaction was stirred at r.t. for 24 h. The
solvent was removed in vacuo and the resulting residue was
partitioned between EtOAc (75 mL) and water (25 mL). The organic
layer was removed, washed with sat. NaHCO.sub.3 solution (25 mL),
then brine (25 mL), and dried (MgSO.sub.4). Removal of the solvent
in vacuo and purification by column chromatography (IH:EtOAc, 5:95,
0:100) afforded the title compound: RT=2.49 min, m/z
(ES.sup.+)=259.2 [M+H].sup.+.
Preparation 11
4-((R)-Cyanomethylmethoxy)piperidine-1-carboxylic acid isopropyl
ester
##STR00028##
[0160] A solution of
4-((R)-1-carbamoylethoxy)piperidine-1-carboxylic acid isopropyl
ester (Preparation 10, 200 mg, 0.78 mmol) in THF, in an oven-dried
flask, under argon, was cooled to 0.degree. C. To the solution was
added triethylamine (320 .mu.L, 2.34 mmol) followed by TFAA (165
.mu.L, 1.16 mmol), and the mixture was stirred at this temperature
for 1 h. The reaction was quenched by the addition of water and the
mixture extracted with DCM (100 mL). The organic fraction was
washed with brine, dried (MgSO.sub.4) and the solvent removed in
vacuo to afford the title compound: RT=3.62 min, m/z
(ES.sup.+)=240.1 [M+H].sup.+.
Preparation 12
4-[(R)-1-(N-Hydroxycarbamimidoyl)ethoxy]piperidine-1-carboxylic
acid isopropyl ester
##STR00029##
[0162] To a solution of
4-((R)-cyanomethylmethoxy)piperidine-1-carboxylic acid isopropyl
ester (Preparation 11, 51 mg, 0.02 mmol) in EtOH (5 mL) was added
hydroxylamine (50% Wt in water, 470 .mu.L, 0.02 mmol) and the
reaction was heated to 80.degree. C. for 16 h. The mixture was
concentrated in vacuo and the residue was azeotroped with toluene
(.times.3) to afford the title compound: RT=2.02 min, m/z
(ES.sup.+)=274.1 [M+H].sup.+.
[0163] The following compounds were prepared by reacting
4-[(R)-1-(N-hydroxycarbamimidoyl)ethoxy]piperidine-1-carboxylic
acid isopropyl ester (Preparation 12) with the appropriate
carboxylic acid, employing the procedure outlined in Preparation
2:
TABLE-US-00002 Prep No. Structure Name LCMS 13 ##STR00030##
4-{(R)-1-[5-(2- Chloropyrimidin-5- yl)-[1,2,4]oxadiazol-
3-yl]ethoxy}- piperidine-1- carboxylic acid isopropyl ester RT =
3.82 min, m/z (ES.sup.+) = 396.1 [M + H].sup.+ 14 ##STR00031##
4-{(R)-1-[5-(5- Chloropyrazin-2-yl)- [1,2,4]oxadiazol-3-
yl]ethoxy}piperidine- 1-carboxylic acid isopropyl ester RT = 3.88
min, m/z (ES.sup.+) = 396.1 [M + H].sup.+
Preparation 15
3-(2,5-Difluorophenyl)-4-nitrobutyric acid methyl ester
##STR00032##
[0165] To a solution of (2E)-3-(2,5-difluorophenyl)acrylic acid
(21.10 g, 114.7 mmol) in a mixture of DCM and MeOH (DCM:MeOH, 4:1,
250 mL) was added a solution of trimethylsilyldiazomethane (2M in
Et.sub.2O, 57.34 mL, 114.7 mmol), over 15 min, and the reaction was
stirred at r.t. until complete. AcOH was added, dropwise, until the
reaction mixture turned colourless, then the solvent was removed in
vacuo. The residue was re-dissolved in MeCN (114 mL), then
nitromethane (7.45 mL, 137.6 mmol) was added. The mixture was
cooled to 0.degree. C. before adding DBU (17.49 mL, 117.0 mmol),
dropwise, over 30 min. The reaction was allowed to warm to r.t. and
stirred for 16 h. Removal of the solvent in vacuo and purification
by column chromatography (IH:EtOAc, 95:5, 90:10) afforded the title
compound: .sup.1H NMR .delta..sub.H (400 MHz, CDCl.sub.3):
7.18-7.00 (m, 3H), 4.91-4.77 (m, 2H), 4.27-4.17 (m, 1H), 3.75 (s,
3H), 2.91 (m, 2H).
Preparation 16
(trans)-1-Benzyl-4-(2,5-difluorophenyl)-5-nitropiperidin-2-one
##STR00033##
[0167] A combination of 3-(2,5-difluorophenyl)-4-nitrobutyric acid
methyl ester (Preparation 15, 16.27 g, 62.81 mmol, paraformaldehyde
(1.94 g, 64.63 mmol) and benzylamine (13.7 mL, 125.62 mmol) in EtOH
was heated to 90.degree. C. in a sealed tube for 16 h. After
complete reaction the mixture was partitioned between EtOAc (400
mL) and 2M HCl (600 mL). The organic fraction was separated, washed
with brine, dried (MgSO.sub.4), and the solvent removed in vacuo.
Purification by column chromatography (IH:EtOAc, 70:30) afforded
the title compound: RT=3.72 min m/z (ES.sup.+)=347.1
[M+H].sup.+.
Preparation 17
(trans)-1-Benzyl-4-(2,5-difluorophenyl)-3-nitropiperidine
hydrochloride HCl
##STR00034##
[0169] To a solution of
(trans)-1-benzyl-4-(2,5-difluorophenyl)-5-nitropiperidin-2-one
(Preparation 16, 10.44 g, 30.17 mmol) in THF (90 mL), under argon,
was added borane dimethylsulfide complex (2.0M in DCM, 45.3 mL,
90.60 mmol) and the reaction was heated to 70.degree. C. for 3 h.
After cooling to r.t. the mixture was diluted with MeOH (20 mL)
then 1M HCl (30 mL) was added. The mixture was stirred for 10 min
before removal of the solvent in vacuo. Further portions of MeOH
(20 mL) and 1M HCl (20 mL) were added and the reaction stirred for
10 min. Removal of the solvent in vacuo afforded the title
compound: RT=3.30 min ink (ES.sup.+)=333.1 [M+H].sup.+.
Preparation 18
[(3R,4R)-1-Benzyl-4-(2,5-difluorophenyl)piperidin-3-yl]carbamic
acid tert-butyl ester
##STR00035##
[0171] A combination of
(trans)-1-benzyl-4-(2,5-difluorophenyl)-3-nitropiperidine
hydrochloride (Preparation 17, 11.12 g, 30.17 mmol) and zinc dust
(15.69 g, 241.36 mmol) in a mixture of AcOH (55 mL) and EtOH (55
mL) was heated to 80.degree. C. After complete reaction the mixture
was filtered and the solvent removed in vacuo. To a solution of the
resulting residue in MeOH (30 mL) was added HCl in dioxane (4M, 30
mL), and the solvent was removed in vacuo. The material was
triturated with Et.sub.2O (.times.2), then toluene (.times.3) to
afford the amine as the hydrochloride salt. To a solution of the
product in a mixture of THF (150 mL) and water (75 mL), cooled to
0.degree. C., was added triethylamine (12.6 mL, 90.51 mmol),
followed by di-tert-butyl dicarbonate (9.59 g, 45.26 mmol). The
reaction was allowed to reach r.t. and stirred for 16 h, until
complete. The mixture was partitioned between EtOAc (750 mL) and
water (200 mL) and the organic phase was separated. The aqueous
phase was extracted with EtOAc (500 mL), then the organic fractions
were combined, dried (MgSO.sub.4) and the solvent removed in vacuo.
Purification by column chromatography (IH:EtOAc, 80:20) and further
purification by chiral HPLC (IH:IPA:DEA 90:10:0.1, 15 ml/min, 270
nm, RT=9.8 min) afforded the title compound: RT=2.68 min m/z
(ES.sup.+)=403.2 [M+H].sup.+.
Preparation 19
[(3R,4R)-4-(2,5-Difluorophenyl)piperidin-3-yl]carbamic acid
tert-butyl ester
##STR00036##
[0173] A solution of
[(3R,4R)-1-benzyl-4-(2,5-difluorophenyl)piperidin-3-yl]carbamic
acid tert-butyl ester (Preparation 18, 1.89 g, 4.70 mmol) in MeOH
(94 mL) was passed through an H-Cube apparatus (10% pd/C Catcart
70, 30 bar, 80.degree. C.) at a flow rate of 1 mL per min. The
solvent was removed in vacuo to afford the title compound: RT=2.37
min; m/z (ES.sup.+)=313.2 [M+H].sup.+.
Preparation 20
2-Methoxypyrimidine-5-carbonitrile
##STR00037##
[0175] To a solution of 5-bromo-2-methoxypyrimidine (2.55 g, 13.49
mmol) and zinc cyanide (1.90 g, 16.19 mmol) in DMF (40 mL), in an
oven-dried flask, was added palladium tetrakis (4.68 g, 4.05 mmol)
and the mixture was bubbled with argon for 10 min before being
heated to 85.degree. C. until complete. The reaction solvent was
concentrated in vacuo and the residue was partitioned between EtOAc
(300 mL) and water (200 mL). The organic layer was separated, then
the aqueous layer was extracted with EtOAc (2.times.150 mL).
Organic fractions were combined, washed with sat. NaHCO.sub.3
solution (2.times.100 mL), brine (100 mL), and dried (MgSO.sub.4).
Removal of the solvent in vacuo and purification by column
chromatography (IH:DCM, 1:9, DCM, DCM:MeOH, 95:5) afforded the
title compound: .sup.1H NMR .delta..sub.H (400 MHz, CDCl.sub.3):
8.80 (s, 2H), 4.12 (s, 3H)
Preparation 21
N-Hydroxy-2-methoxypyrimidine-5-carboxamidine
##STR00038##
[0177] To a solution of 2-methoxypyrimidine-5-carbonitrile
(Preparation 20, 1.21 g, 9.01 mmol) in EtOH (50 mL) was added
hydroxylamine (50% Wt in water, 0.65 mL, 9.91 mmol) and the
reaction was heated to 65.degree. C. for 16 h. The mixture was
concentrated in vacuo to afford the title compound: RT=0.85 min,
m/z (ES.sup.+)=169.0 [M+H].sup.+.
Preparation 22
4-[3-(2-Methoxypyrimidin-5-yl)-[1,2,4]oxadiazol-5-ylmethoxy]piperidine-1-c-
arboxylic acid tert-butyl ester
##STR00039##
[0179] To a solution of 4-carboxymethoxypiperidine-1-carboxylic
acid tert-butyl ester (1.08 g, 4.16 mmol) in THF (30 mL) was added
EDCI (0.96 g, 5.00 mmol) followed by HOBt (0.77 g, 5.00 mmol), and
the reaction was stirred at r.t. for 15 min.
N-Hydroxy-2-methoxypyrimidine-5-carboxamidine (Preparation 21, 0.70
g, 4.16 mmol) was added and the reaction was stirred at r.t. for 16
h before removing the solvent in vacuo. The resulting residue was
partitioned between EtOAc (50 mL) and water (50 mL), then the
organic phase was separated, washed with sat. NaHCO.sub.3 solution
(50 mL), brine (50 mL), and dried (MgSO.sub.4), before removal of
the solvent in vacuo. The residue was dissolved in toluene (30 mL)
and heated to 85.degree. C. until completion. Removal of the
solvent in vacuo afforded the title compound: RT=3.58 min, m/z
(ES.sup.+)=392.2 [M+H].sup.+.
Preparation 23
4-[3-(2-Methoxypyrimidin-5-yl)-[1,2,4]oxadiazol-5-ylmethoxy]piperidine-1-c-
arboxylic acid isopropyl ester
##STR00040##
[0181] To a solution of
4-[3-(2-methoxypyrimidin-5-yl)-[1,2,4]oxadiazol-5-ylmethoxy]piperidine-1--
carboxylic acid tert-butyl ester (Preparation 22, 1.33 g, 3.40
mmol) in dioxane (10 mL) was added HCl in dioxane (4M, 2.55 mL,
10.19 mmol) and the reaction was stirred at r.t. for 4 h. A further
portion of HCl in dioxane (7.5 mL, 30 mmol) was added and stirring
continued for 72 h. Removal of the solvent in vacuo afforded the
intermediate product
2-methoxy-5-[5-(piperidin-4-yloxymethyl)-[1,2,4]oxadiazol-3-yl]pyrimidine
hydrochloride: RT=1.95 min, m/z (ES.sup.+)=292.1 [M+H].sup.+.
[0182] To a suspension of the product in DCM (20 mL) was added
triethylamine (0.99 mL, 7.11 mmol) and the mixture was cooled to
0.degree. C. Isopropyl chloroformate (1M in toluene, 3.73 mL, 3.73
mmol) was added, dropwise, over 20 min, and the reaction was
allowed to stir for a further 2 h. The mixture was partitioned
between DCM (50 mL) and water (50 mL) and the organic phase was
separated. The aqueous phase was extracted with DCM (100 mL) then
organic fractions were combined, washed with brine (100 mL), dried
(MgSO.sub.4), and the solvent removed in vacuo. Purification by
column chromatography (DCM:MeOH, 98:2, 97:3, 96:4) afforded the
title compound: RT=3.44 min, m/z (ES.sup.+)=378.2 [M+H].sup.+.
Preparation 24
4-[3-(2-Hydroxypyrimidin-5-yl)-[1,2,4]oxadiazol-5-ylmethoxy]piperidine-1-c-
arboxylic acid isopropyl ester
##STR00041##
[0184] To a solution of
4-[3-(2-methoxypyrimidin-5-yl)-[1,2,4]oxadiazol-5-ylmethoxy]piperidine-1--
carboxylic acid isopropyl ester (Preparation 23, 885 mg, 2.34 mmol)
in MeCN (30 mL), under an atmosphere of argon, was added sodium
iodide (1054 mg, 7.03 mmol) followed by trimethylsilyl chloride
(893 .mu.L, 7.03 mmol), and the reaction was heated to 65.degree.
C. for 16 h. The mixture was partitioned between EtOAc (100 mL) and
sat. sodium thiosulfate solution (100 mL), and the organic phase
was separated. The aqueous phase was extracted with EtOAc
(2.times.50 mL) then organic fractions were combined, washed with
brine (100 mL), dried (MgSO.sub.4), and the solvent removed in
vacuo. Purification by column chromatography (DCM:MeOH, 100:0,
98:2, 96:4, 94:6) afforded the title compound: RT=2.76 min, m/z
(ES.sup.+)=364.2 [M+H].sup.+.
Preparation 25
4-[3-(2-Chloropyrimidin-5-yl)-[1,2,4]oxadiazol-5-ylmethoxy]piperidine-1-ca-
rboxylic acid isopropyl ester
##STR00042##
[0186] To a solution of
4-[3-(2-hydroxypyrimidin-5-yl)-[1,2,4]oxadiazol-5-ylmethoxy]piperidine-1--
carboxylic acid isopropyl ester (Preparation 24, 199 mg, 0.55 mmol)
in phosphorus oxychloride (6 mL), under argon, was added
N,N-dimethylaminoaniline (90 .mu.L, 0.71 mmol) and the mixture was
heated to 50.degree. C. for 5 h. The reaction was quenched by being
added, dropwise, to ice (50 mL) and the organics were extracted
into DCM (3.times.50 mL). The organic fractions were combined,
washed with brine (50 mL), dried (MgSO.sub.4), and the solvent
removed in vacuo. Purification by column chromatography (DCM:MeOH,
97:3) afforded the title compound: RT=3.71 min, m/z
(ES.sup.+)=382.1 [M+H].sup.+.
Preparation 26
4-{(R)-1-[3-(2-Methoxypyrimidin-5-yl)-[1,2,4]oxadiazol-5-yl]ethoxy}piperid-
ine-1-carboxylic acid isopropyl ester
##STR00043##
[0188] 4-((R)-1-Carboxyethoxy)piperidine-1-carboxylic acid
isopropyl ester (Preparation 9) was reacted with
N-hydroxy-2-methoxypyrimidine-5-carboxamidine (Preparation 21)
employing the procedure outlined in Preparation 22. Purification by
column chromatography (DCM:MeOH, 97:3) afforded the title compound:
RT=3.55 min, m/z (ES.sup.+)=392.2 [M+H].sup.+.
Preparation 27
4-{(R)-1-[3-(2-Hydroxypyrimidin-5-yl)-[1,2,4]oxadiazol-5-yl]ethoxy}piperid-
ine-1-carboxylic acid isopropyl ester
##STR00044##
[0190] The title compounds was prepared from
4-{(R)-1-[3-(2-methoxypyrimidin-5-yl)-[1,2,4]oxadiazol-5-yl]ethoxy}piperi-
dine-1-carboxylic acid isopropyl ester (Preparation 26) employing
the procedure outlined in Preparation 24: RT=2.87 min, m/z
(ES.sup.+)=378.2 [M+H].sup.+.
Preparation 28
4-{(R)-1-[3-(2-Chloropyrimidin-5-yl)-[1,2,4]oxadiazol-5-yl]ethoxy}-piperid-
ine-1-carboxylic acid isopropyl ester
##STR00045##
[0192] The title compound was prepared from
4-{(R)-1-[3-(2-hydroxypyrimidin-5-yl)-[1,2,4]oxadiazol-5-yl]ethoxy}piperi-
dine-1-carboxylic acid isopropyl ester (Preparation 27) employing
the procedure outlined in Preparation 25: RT=3.82 min, m/z
(ES.sup.+)=396.1 [M+H].sup.+.
Preparation 29
(trans)-3-(9H-Fluoren-9-ylmethoxycarbonylamino)-4-(2-fluorophenyl)pyrrolid-
ine-1-carboxylic acid tert-butyl ester
##STR00046##
[0194] To a solution of
(trans)-3-amino-4-(2-fluorophenyl)pyrrolidine-1-carboxylic acid
tert-butyl ester (2.00 g, 7.13 mmol) and triethylamine (1.59 mL,
11.40 mmol) in a combination of dioxane and water (2:1, 75 mL),
cooled to 0.degree. C., was added 9-fluorenylmethyl chloroformate
(2.31 g, 8.92 mmol). The reaction was allowed to reach r.t. before
stirring for 16 h. The mixture was diluted with EtOAc, then the
solution was washed with water, 1M HCl, sat. NaHCO.sub.3 solution,
brine, and dried (MgSO.sub.4). Removal of the solvent in vacuo and
purification by column chromatography (IH:EtOAc, 90:10, 80:20,
70:30) afforded the title compound: RT=4.28 min m/z
(ES.sup.+)=503.3 [M+H].sup.+.
Preparation 30
[(trans)-4-(2-Fluorophenyl)pyrrolidin-3-yl]carbamic acid
9H-fluoren-9-ylmethyl ester hydrochloride
##STR00047##
[0196] To a solution of
(trans)-3-(9H-fluoren-9-ylmethoxycarbonylamino)-4-(2-fluorophenyl)pyrroli-
dine-1-carboxylic acid tert-butyl ester (Preparation 29, 1.50 g,
2.98 mmol) in dioxane (30 mL) was added a solution of HCl in
dioxane (4M, 30 mL) and the reaction was stirred at r.t. for 16 h,
after which time a precipitate had formed. Et.sub.2O was added to
the mixture until no further precipitation was observed, and the
solvent was decanted. The residue was suspended in a further volume
of Et.sub.2O and the mixture was stirred for 5 min before decanting
the solvent. This process was repeated twice more and the resulting
residue was concentrated to dryness to afford the title compound:
RT=2.82 min m/z (ES.sup.+)=403.1 [M+H].sup.+.
Preparation 31
(3R,4S)-3-(9H-Fluoren-9-ylmethoxycarbonylamino)-4-(2-fluorophenyl)pyrrolid-
ine-1-carboxylic acid tert-butyl ester
##STR00048##
[0198] The title compound was afforded via chiral HPLC separation
of
(trans)-3-(9H-fluoren-9-ylmethoxycarbonylamino)-4-(2-fluorophenyl)pyrroli-
dine-1-carboxylic acid tert-butyl ester (Preparation 29):
IH:CHCl.sub.3:IPA:DEA 85:10:5:0.1, 15 mL/min, 270 nm, RT=9.4
min.
Preparation 32
[(3R,4S)-4-(2-Fluorophenyl)pyrrolidin-3-yl]carbamic acid
9H-fluoren-9-ylmethyl ester hydrochloride
##STR00049##
[0200] The title compound was prepared from
(3R,4S)-3-(9H-fluoren-9-ylmethoxycarbonylamino)-4-(2-fluorophenyl)pyrroli-
dine-1-carboxylic acid tert-butyl ester (Preparation 31) employing
the procedure outlined in Preparation 30: RT=2.82 min m/z
(ES.sup.+)=403.1 [M+H].sup.+.
Preparation 33
2,4-Difluoro-1-((E)-2-nitrovinyl)benzene
##STR00050##
[0202] To a solution of 2,4-difluorobenzaldehyde (25.0 g, 0.18 mol)
and nitromethane (11.4 mL, 0.21 mol) in MeOH (53 mL), under argon,
cooled to -15.degree. C., was added a solution of NaOH (7.4 g, 0.19
mol) in water (26 mL), dropwise, over 20 min. The resulting mixture
was stirred at -15.degree. C. and a precipitate formed after 30
min. More MeOH was added to form a slurry and stirring continued
for 15 min before allowing the reaction to warm to 0.degree. C. Ice
water was added and the mixture was stirred for 15 min before
adding 4M HCl (100 mL). The organic fraction was extracted with DCM
(3.times.300 mL), dried (Na.sub.2SO.sub.4) and the solvent removed
in vacuo. A portion of the residue (10.0 g, 50 mmol) was dissolved
in acetic anhydride (8.1 mL, 90 mol) and cooled to 0.degree. C.
under argon. DMAP (0.4 g, 3 mmol) was added and the reaction was
stirred at this temperature for 20 min before warming the mixture
to r.t. and allowing it to stir for a further 16 h. The reaction
solvent was removed in vacuo and the resulting residue was
re-dissolved in DCM. Remaining acetic anhydride was destroyed by
the addition of a small volume of 1M NaOH solution, then the
resulting solution was dried (MgSO.sub.4) and concentrated in
vacuo. Purification by column chromatography (DCM) afforded the
title compound: RT=3.60 min; m/z (ES.sup.+)=186.1 [M+H].sup.+.
Preparation 34
(trans)-1-Benzyl-3-(2,4-difluorophenyl)-4-nitropyrrolidine
##STR00051##
[0204] A solution of 2,4-difluoro-1-((E)-2-nitrovinyl)benzene
(Preparation 33, 8.0 g, 43.0 mmol) in DCM (250 mL), under argon,
was cooled to -30.degree. C.
N-(Methoxymethyl)-N-(trimethylsilylmethyl)benzylamine (11.7 mL,
45.0 mmol) was added so as to maintain the temperature at
-30.degree. C. The reaction was stirred for 10 min before the
dropwise addition of TFA (0.3 mL, 4.3 mmol), and the resulting
mixture was allowed to stir at r.t. over 16 h. The reaction mixture
was washed with water, then brine, and dried (Na.sub.2SO.sub.4).
Removal of the solvent in vacuo to afford the title compound:
RT=3.05 min; m/z (ES.sup.+)=319.1 [M+H].sup.+.
Preparation 35
[(trans)-1-Benzyl-4-(2,4-difluorophenyl)pyrrolidin-3-yl]carbamic
acid tert-butyl ester
##STR00052##
[0206] A combination of
(trans)-1-benzyl-3-(2,4-difluorophenyl)-4-nitropyrrolidine
(Preparation 34, 25.0 g, 0.08 mol) and zinc dust (17.8 g, 0.28 mol)
in a mixture of AcOH and EtOH (1:1, 500 mL) was heated to
70.degree. C. After 45 h a further portion of zinc dust (12.0 g,
0.18 mol) was added and heating continued for 20 min. After
complete reaction the solvent was removed in vacuo. The resulting
residue was re-dissolved in EtOAc, washed with sat. NaHCO.sub.3
solution, then brine, and dried (Na.sub.2SO.sub.4). Removal of the
solvent in vacuo afforded the intermediate product
(trans)-1-benzyl-4-(2,4-difluorophenyl)pyrrolidin-3-ylamine:
RT=1.82 min; m/z (ES.sup.+)=289.1 [M+H].sup.+. To a solution of the
product in THF (400 mL), under argon, was added triethylamine (20.4
mL, 0.15 mol) and the solution was cooled to 0.degree. C.
Di-tert-butyl dicarbonate (19.0 g, 0.09 mol) was added over 5 min,
and the reaction was allowed to reach r.t. over 16 h. The solvent
was removed in vacuo, then the resulting residue was re-dissolved
in EtOAc, washed with brine, dried (Na.sub.2SO.sub.4), and the
solvent removed in vacuo. To the product was added heptane (100
mL), and the suspension was sonicated until fully dissolved. The
solution was allowed to stand for 60 h, allowing formation of a
precipitate. The solvent was decanted and the solids were washed
with a fresh portion of heptane (50 mL) to afford the title
compound: RT=2.74 min; m/z (ES.sup.+)=389.3 [M+H].sup.+.
Preparation 36
[(3R,4S)-1-Benzyl-4-(2,4-difluorophenyl)pyrrolidin-3-yl]carbamic
acid tert-butyl ester
##STR00053##
[0208] The title compound was afforded via chiral HPLC separation
of [(trans)-1-benzyl-4-(2,4-difluorophenyl)pyrrolidin-3-yl]carbamic
acid tert-butyl ester (Preparation 35): IH:IPA:DEA 96:4:0.1, 15
mL/min, 270 nm, RT=9.8 min.
Preparation 37
[(3R,4S)-4-(2,4-Difluorophenyl)pyrrolidin-3-yl]carbamic acid
tert-butyl ester
##STR00054##
[0210] The title compound was prepared from
[(3R,4S)-1-benzyl-4-(2,4-difluorophenyl)pyrrolidin-3-yl]carbamic
acid tert-butyl ester (Preparation 36) employing the procedure
outlined in Preparation 19, but at a pressure of 10 bar and
temperature of 50.degree. C.: RT=2.38 min; m/z (ES.sup.+)=299.1
[M+H].sup.+.
Preparation 38
4-((R)-1-{5-[6-(Benzotriazol-1-yloxy)pyridazin-3-yl]-[1,2,4]oxadiazol-3-yl-
}ethoxy)piperidine-1-carboxylic acid isopropyl ester
##STR00055##
[0212] To a solution of 6-chloropyridazine-3-carboxylic acid (60
mg, 0.38 mmol) in THF (10 mL) was added EDCI (107 mg, 0.46 mmol),
followed HOBt (69 mg, 0.46 mmol), and the reaction was stirred at
r.t. for 15 min
4-[(R)-1-(N-Hydroxycarbamimidoyl)ethoxy]piperidine-1-carboxylic
acid isopropyl ester (Preparation 12, 103 mg, 0.38 mmol) was added
and stirring continued for 4 h, before removing the solvent in
vacuo. The resulting residue was partitioned between EtOAc and
water, then the organic phase was separated, washed with sat.
NaHCO.sub.3 solution, dried (MgSO.sub.4) and the solvent removed in
vacuo. The residue was dissolved in toluene and heated to reflux
for 16 h before concentrating the solvent in vacuo and redissolving
the product in EtOAc. The solution was washed with water, then
brine, and dried (MgSO.sub.4), before removal of the solvent in
vacuo. Purification by column chromatography (IH:EtOAc, 1:1)
afforded the title compound: RT=3.87 min, m/z (ES.sup.+)=495.2
[M+H].sup.+.
Preparation 39
3-((R)-1-Carboxyethoxy)azetidine-1-carboxylic acid tert-butyl
ester
##STR00056##
[0214] A solution of 3-hydroxyazetidine-1-carboxylic acid
tert-butyl ester (3.00 g, 17.32 mmol) in THF (50 mL), under argon,
was cooled to 0.degree. C. and sodium hydride (60% in mineral oil,
0.76 g, 18.98 mmol) was added, portionwise, over 10 min. The
reaction was stirred at r.t. for 72 h, then water (50 mL) was
added. The mixture was washed with EtOAc (2.times.100 mL) and the
resulting aqueous solution was acidified to pH1 with 2M HCl. The
mixture was extracted with EtOAc (3.times.80 mL) then organic
fractions were combined, washed with brine (100 mL), dried
(MgSO.sub.4) and the solvent removed in vacuo to afford the title
compound: .sup.1H NMR .delta..sub.H (400 MHz, CDCl.sub.3):
4.40-4.31 (m, 1H), 4.16-4.08 (m, 2H), 4.04-3.93 (m, 2H), 3.90-3.85
(m, 1H), 1.48 (d, T=7.0 Hz, 3H), 1.44 (s, 9H).
Preparation 40
3-((R)-1-Methoxycarbonylethoxy)azetidine-1-carboxylic acid
tert-butyl ester
##STR00057##
[0216] To a solution of
3-((R)-1-carboxyethoxy)azetidine-1-carboxylic acid tert-butyl ester
(Preparation 39, 3.29 g, 13.40 mmol) in a mixture of toluene and
MeOH (4:1, 25 mL), cooled to 0.degree. C., was added
trimethylsilyldiazomethane (2M in hexanes, 8.71 mL, 17.42 mmol),
dropwise, over 5 min. The reaction was stirred at this temperature
for 30 min before being quenched by the addition of AcOH (1 mL).
Removal of the solvent in vacuo and purification by column
chromatography (DCM:MeOH, 100:0, 98:2, 95:5) afforded the title
compound: .sup.1H NMR .delta..sub.H (400 MHz, CDCl.sub.3):
4.35-4.27 (m, 1H), 4.13-4.04 (m, 2H), 3.99-3.91 (m, 2H), 3.87-3.82
(m, 1H), 3.75 (s, 3H), 1.44-1.41 (m, 12H).
Preparation 41
(R)-2-(Azetidin-3-yloxy)propionic acid methyl ester trifluoroacetic
acid salt
##STR00058##
[0218] To a solution of
3-((R)-1-methoxycarbonylethoxy)azetidine-1-carboxylic acid
tert-butyl ester (Preparation 40, 2.69 g, 10.37 mmol) in DCM (50
mL), under argon, cooled to 0.degree. C., was added TFA (10 mL) and
the reaction was stirred at this temperature for 1 h. A further
portion of TFA (2 mL) was added and stirring continued for 30 min.
Removal of the solvent in vacuo afforded the title compound:
.sup.1H NMR .delta..sub.H (400 MHz, DMSO-d.sub.6): 4.51-4.43 (m,
1H), 4.24-4.17 (m, 1H), 4.16-4.08 (m, 2H), 3.96-3.83 (m, 2H), 3.66
(s, 3H), 1.30 (d, J=6.6 Hz, 3H).
Preparation 42
3-((R)-1-Methoxycarbonylethoxy)azetidine-1-carboxylic acid
isopropyl ester
##STR00059##
[0220] A suspension of (R)-2-(azetidin-3-yloxy)propionic acid
methyl ester trifluoroacetic acid salt (Preparation 41, 1.42 g,
5.19 mmol) in DCM (20 mL), under argon, was cooled to 0.degree. C.
Triethylamine (1.66 mL, 11.93 mmol) was added, followed by a
solution of isopropyl chloroformate (1M in toluene, 6.22 mL, 6.22
mmol), dropwise, and the reaction was stirred at this temperature
for 2 h. The mixture was diluted with DCM (30 mL), washed with 1M
HCl (30 mL), sat. NaHCO.sub.3 solution (30 mL), then brine (30 mL),
and dried (MgSO.sub.4). Removal of the solvent in vacuo and
purification by column chromatography (DCM:MeOH, 99:0, 98:2)
afforded the title compound: .sup.1H NMR .delta..sub.H (400 MHz,
CDCl.sub.3): 4.94-4.86 (m, 1H), 4.38-4.30 (m, 1H), 4.17-4.09 (m,
2H), 4.01-3.94 (m, 2H), 3.91-3.86 (m, 1H), 3.76 (s, 3H), 1.43 (d,
J=7.0 Hz, 3H), 1.22 (d, J=6.2 Hz, 6H).
Preparation 43
3-((R)-1-Carboxyethoxy)azetidine-1-carboxylic acid isopropyl
ester
##STR00060##
[0222] To a solution of
3-((R)-1-methoxycarbonylethoxy)azetidine-1-carboxylic acid
isopropyl ester (Preparation 42, 829 mg, 3.38 mmol) in THF (6 mL),
cooled to 0.degree. C., was added water (1.5 mL) followed by
lithium hydroxide monohydrate (298 mg, 7.10 mmol), and the
resulting mixture was stirred at this temperature for 3 h. The
reaction solvent was concentrated in vacuo, then water (15 mL) was
added and the solution washed with EtOAc (20 mL). The aqueous phase
was acidified to pH 1 with 1M HCl and extracted with EtOAc
(2.times.30 mL). The organic fractions were combined, washed with
water (20 mL), dried (MgSO.sub.4), and the solvent removed in vacuo
to afford the title compound: .sup.1H NMR .delta..sub.H (400 MHz,
DMSO-d.sub.6): 4.77-4.66 (m, 1H), 4.39-4.31 (m, 1H), 4.08-4.00 (m,
2H), 3.99-3.92 (m, 1H), 3.77-3.66 (m, 2H), 1.28 (d, J=6.6 Hz, 3H),
1.15 (d, 6H).
Preparation 44
2,5-Difluoro-1-((E)-2-nitrovinyl)benzene
##STR00061##
[0224] The title compound was prepared from
2,5-difluorobenzaldehyde employing a similar method to that
outlined in Preparation 33. After reaction with DMAP the crude
mixture was diluted with sat. NaHCO.sub.3 solution. The precipitate
that formed was stirred for 30 min, filtered, and dried to afford
the title compound: .sup.1H NMR .delta..sub.H (300 MHz,
CDCl.sub.3): 8.00-7.96 (m, 1H), 7.71-7.66 (m, 1H), 7.25-7.11 (m,
3H).
Preparation 45
(trans)-1-Benzyl-3-(2,5-difluorophenyl)-4-nitropyrrolidine
##STR00062##
[0226] The title compound was prepared from
2,5-difluoro-1-((E)-2-nitrovinyl)benzene (Preparation 44) employing
the method outlined in Preparation 34, but the reaction was carried
out at 0.degree. C. Purification by column chromatography
(Hexane:EtOAc, 100:0, 98:2, 95:5, 90:10) afforded the title
compound. LCMS method 2: RT=0.96 min; m/z (ES.sup.+)=319.2
[M+H].sup.+.
Preparation 46
[(trans)-1-Benzyl-4-(2,5-difluorophenyl)pyrrolidin-3-yl]carbamic
acid tert-butyl ester
##STR00063##
[0228] A combination of
(trans)-1-benzyl-3-(2,5-difluorophenyl)-4-nitropyrrolidine
(Preparation 45, 45.6 g, 0.14 mol) and zinc dust (57.0 g, 0.86 mol)
in a mixture of AcOH and EtOH (1:1, 820 mL) was heated to
65.degree. C. After complete reaction the mixture was filtered,
washing with AcOH, and the filtrate was concentrated in vacuo. The
resulting residue was re-dissolved in EtOAc, washed with sat.
NaHCO.sub.3 solution, then brine, and dried (Na.sub.2SO.sub.4).
Removal of the solvent in vacuo and purification by column
chromatography (DCM:MeOH:NH.sub.3, 100:0:0, 95:5:0, 90:10:0,
90:10:5) afforded the intermediate product
(trans)-1-benzyl-4-(2,4,5-trifluorophenyl)pyrrolidin-3-ylamine LCMS
method 2: RT=0.80 min; m/z (ES.sup.+)=289.4 [M+H].sup.+. To a
solution of the product (12.2 g, 42.3 mmol) in THF (250 mL), under
argon, was added triethylamine (12.0 mL, 84.6 mol) and the mixture
was cooled to 0.degree. C. Di-tert-butyl dicarbonate (11.0 g, 50.4
mol) was added over 5 min, then the reaction was allowed to reach
r.t. and stirred for 16 h. The solvent was concentrated, then the
resulting residue was re-dissolved in EtOAc, washed with brine,
dried (Na.sub.2SO.sub.4), and the solvent removed in vacuo. The
product was triturated several times with heptane to afford the
title compound. LCMS method 3: RT=3.08 min; m/z (ES.sup.+)=389.5
[M+H].sup.+.
Preparation 47
[(3R,4S)-1-Benzyl-4-(2,5-difluorophenyl)pyrrolidin-3-yl]carbamic
acid tert-butyl ester
##STR00064##
[0230] The title compound was afforded via chiral HPLC separation
of [(trans)-1-benzyl-4-(2,5-difluorophenyl)pyrrolidin-3-yl]carbamic
acid tert-butyl ester (Preparation 46): IH:IPA:DEA 96:4:0.1, 15
mL/min, 270 nm, RT=10.9 min.
Preparation 48
[(3R,4S)-4-(2,5-Difluorophenyl)pyrrolidin-3-yl]carbamic acid
tert-butyl ester
##STR00065##
[0232] The title compound was prepared from
[(3R,4S)-1-benzyl-4-(2,5-difluorophenyl)pyrrolidin-3-yl]carbamic
acid tert-butyl ester (Preparation 47) employing the procedure
outlined in Preparation 19, but the reaction was carried out at
90.degree. C. RT=2.35 min; m/z (ES.sup.+)=299.2 [M+H].sup.+.
Preparation 49
[(3R,4S)-1-(5-Cyanopyrimidin-2-yl)-4-(2,5-difluorophenyl)pyrrolidin-3-yl]c-
arbamic acid tert-butyl ester
##STR00066##
[0234] To a mixture of 2-chloropyrimidine-5-carbonitrile (71 mg,
0.51 mmol) and
[(3R,4S)-4-(2,5-difluorophenyl)pyrrolidin-3-yl]carbamic acid
tert-butyl ester (Preparation 48, 160 mg, 0.54 mmol) in DMSO (1 mL)
was added DBU (76 .mu.L, 0.51 mmol). The mixture was bubbled with
argon for 5 min and then heated to 70.degree. C. for 16 h. After
cooling to r.t. the crude reaction mixture was partitioned between
EtOAc (50 mL) and water (50 mL), and the organic phase was
separated. The aqueous phase was extracted further with EtOAc (20
mL) then the organic fractions were combined, washed with water (30
mL), sat. NaHCO.sub.3 solution (50 mL), then brine (50 mL), and
dried (MgSO.sub.4). Removal of the solvent in vacuo and
purification by column chromatography (DCM:MeOH, 100:0, 98:2)
afforded the title compound: RT=4.03 min; m/z (ES.sup.+)=402.1
[M+H].sup.+.
Preparation 50
{(3R,4S)-4-(2,5-Difluorophenyl)-1-[5-(N-hydroxycarbamimidoyl)-pyrimidin-2--
yl]pyrrolidin-3-yl}carbamic acid tert-butyl ester
##STR00067##
[0236] The title compound was prepared from
[(3R,4S)-1-(5-cyanopyrimidin-2-yl)-4-(2,5-difluorophenyl)pyrrolidin-3-yl]-
carbamic acid tert-butyl ester (Preparation 49) employing the
procedure outlined in Preparation 21: RT=2.72 min; m/z
(ES.sup.+)=435.2 [M+H].sup.+.
Preparation 51
3-[(R)-1-(3-{2-[(3R,4S)-3-tert-Butoxycarbonylamino-4-(2,5-difluorophenyl)p-
yrrolidin-1-yl]pyrimidin-5-yl}-[1,2,4]oxadiazol-5-yl)ethoxy]azetidine-1-ca-
rboxylic acid isopropyl ester
##STR00068##
[0238] 3-((R)-1-Carboxyethoxy)azetidine-1-carboxylic acid isopropyl
ester (Preparation 43) was reacted with
{(3R,4S)-4-(2,5-difluorophenyl)-1-[5-(N-hydroxycarbamimidoyl)pyrimidin-2--
yl]pyrrolidin-3-yl}carbamic acid tert-butyl ester (Preparation 50)
employing the procedure outlined in Preparation 22. Purification by
column chromatography (IH:EtOAc, 6:4) afforded the title compound:
RT=4.44 min, m/z (ES.sup.+)=630.2 [M+H].sup.+.
Preparation 52
[(3R,4S)-1-(5-Cyanopyrazin-2-yl)-4-(2,5-difluorophenyl)pyrrolidin-3-yl]car-
bamic acid tert-butyl ester
##STR00069##
[0240] To a mixture of 5-chloropyrazine-2-carbonitrile (140 mg,
1.01 mmol) and
[(3R,4S)-4-(2,5-difluorophenyl)pyrrolidin-3-yl]carbamic acid
tert-butyl ester (Preparation 48, 300 mg, 1.01 mmol) in DMSO (1 mL)
was added DBU (150 .mu.L, 1.01 mmol). The mixture was bubbled with
argon for 5 min and then heated to 70.degree. C. for 3 h. After
cooling to r.t. the crude reaction mixture was partitioned between
EtOAc (200 mL) and water (75 mL), then the organic phase was
separated, washed with brine, dried (MgSO.sub.4), and the solvent
removed in vacuo. Purification by column chromatography (IH:EtOAc,
60:40) afforded the title compound: RT=3.93 min; m/z
(ES.sup.+)=402.1 [M+H].sup.+.
Preparation 53
{(3R,4S)-4-(2,5-Difluorophenyl)-1-[5-(N-hydroxycarbamimidoyl)pyrazin-2-yl]-
pyrrolidin-3-yl}carbamic acid tert-butyl ester
##STR00070##
[0242] The title compound was prepared from
[(3R,4S)-1-(5-cyanopyrazin-2-yl)-4-(2,5-difluorophenyl)pyrrolidin-3-yl]ca-
rbamic acid tert-butyl ester (Preparation 52) employing the
procedure outlined in Preparation 12, but the reaction was only
heated for 3 h: RT=2.73 min; m/z (ES.sup.+)=435.2 [M+H].sup.+.
Preparation 54
[(trans)-1-Benzyl-4-(2,4,5-trifluorophenyl)pyrrolidin-3-yl]carbamic
acid tert-butyl ester
##STR00071##
[0244] The title compound was prepared in 3 steps from
2,4,5-trifluorobenzaldehyde employing the procedures outlined in
the synthesis of Preparation 46. LCMS method 3: RT=3.10 min; m/z
(ES.sup.+)=407.3 [M+H].sup.+.
Preparation 55
[(trans)-4-(2,4,5-Trifluorophenyl)pyrrolidin-3-yl]carbamic acid
tert-butyl ester
##STR00072##
[0246] A solution of
[(trans)-1-benzyl-4-(2,4,5-trifluorophenyl)pyrrolidin-3-yl]carbamic
acid tert-butyl ester (Preparation 54, 40.1 g, 98.8 mmol) in a
combination of IMS (325 mL) and EtOAc (50 mL), in an autoclave, was
placed under an atmosphere of argon. Palladium on carbon (5%, 4.0
g, 1.9 mmol) was added as a slurry in the minimum volume of
toluene, then the reaction mixture was placed under an atmosphere
of hydrogen (50 atm) and stirred for 72 h. The crude mixture was
filtered through celite, washing with EtOAc, and the filtrate was
concentrated in vacuo to afford the title compound. LCMS method 4:
RT=2.42 min; ink (ES.sup.+)=317.2 [M+H].sup.+.
Preparation 56
[(3R,4S)-4-(2,4,5-Trifluorophenyl)pyrrolidin-3-yl]carbamic acid
tert-butyl ester
##STR00073##
[0248] [(trans)-4-(2,4,5-Trifluorophenyl)pyrrolidin-3-yl]carbamic
acid tert-butyl ester (Preparation 55, 59.5 g, 188 mmol) was
suspended in EtOH (200 mL) and heated to 70.degree. C. To the
suspension was added a warm solution of (S)-(+)-naproxen (21.5 g,
93 mmol) and the mixture was heated to reflux. The heat was removed
and the mixture slowly allowed to cool to r.t., with stirring, for
16 h. The resulting precipitate was filtered, washing with EtOH,
and the filtrate was partitioned between DCM (2400 mL) and 1M NaOH
(600 mL). The organic phase was separated, washed with 1M NaOH,
brine, then dried (MgSO.sub.4), and the solvent was removed in
vacuo. The whole process was repeated for a second time to afford
the title compound: .sup.1H NMR .delta..sub.H (400 MHz,
CD.sub.3OD): 7.38-7.25 (m, 1H), 7.14-7.01 (m, 1H).
Preparation 57
3-Hydroxypyrrolidine-1-carboxylic acid isopropyl ester
##STR00074##
[0250] A solution of 3-hydroxypyrrolidine-1-carboxylic acid
tert-butyl ester (10.0 g, 53.4 mmol) in a solution of HCl in
dioxane (4M, 125 mL) was stirred at r.t. for 40 min before removal
of the reaction solvent in vacuo. The residue was suspended in DCM
(150 mL), and triethylamine (22.0 mL, 160.2 mmol) was added before
cooling the reaction mixture to 0.degree. C. A solution of
isopropyl chloroformate in toluene (1M, 64 mL, 64 mmol) was added,
dropwise, then the resulting mixture was removed from the ice bath
and stirred at r.t. for 1 h. A further portion of isopropyl
chloroformate (53.4 ml, 53.4 mmol) was added and stirring continued
at r.t. for 16 h. The crude reaction mixture was washed with water
(2.times.100 mL), then dried (MgSO.sub.4). Removal of the solvent
in vacuo and purification by column chromatography
(DCM:MeOH:NH.sub.4OH, 9:1:0.1) afforded the title compound: RT=2.29
min; m/z (ES.sup.+)=174.0 [M+H].sup.+.
Preparation 58
3-((R)-1-Carboxyethoxy)pyrrolidine-1-carboxylic acid isopropyl
ester
##STR00075##
[0252] The title compound was prepared employing a similar method
to that outlined in Preparation 9. After initial work-up the
residue was dissolved in 1M NaOH solution, and washed with
Et.sub.2O. The aqueous phase was acidified to pH1 with 2M HCl,
extracted with EtOAc, and the organic phase dried
(Na.sub.2SO.sub.4). Removal of the solvent in vacuo afforded the
title compound: RT=2.70 min; m/z (ES.sup.+)=246.2 [M+H].sup.+.
Preparation 59
3-((R)-1-Carbamoylethoxy)pyrrolidine-1-carboxylic acid isopropyl
ester
##STR00076##
[0254] To a solution of
3-((R)-1-carboxyethoxy)pyrrolidine-1-carboxylic acid isopropyl
ester (Preparation 58, 1.00 g, 4.08 mmol) in THF (50 mL), under
argon, was added EDCI (0.94 g, 4.90 mmol), followed by HOBt (0.66
g, 4.33 mmol), and the reaction was stirred at r.t. for 10 min. A
solution of NH.sub.3 in dioxane (0.05M, 20.4 .mu.L, 40.80 mmol) was
added and the reaction was stirred at r.t. for 4 h. A further
portion of NH.sub.3 in dioxane (0.05M, 10 mL) was added and the
reaction was stirred for 1 h. The solvent was removed in vacuo and
the resulting residue was partitioned between EtOAc (150 mL) and
water (100 mL). The organic phase was removed, and the aqueous
phase was extracted with EtOAc (150 mL). The organic fractions were
combined, washed with sat. NaHCO.sub.3 solution (100 mL), then
brine (100 mL), and dried (MgSO.sub.4). Removal of the solvent in
vacuo and purification by column chromatography (DCM:MeOH, 95:5)
afforded the title compound: RT=2.53 min, m/z (ES.sup.+)=245.2
[M+H].sup.+.
Preparation 60
3-((R)-Cyanomethylmethoxy)pyrrolidine-1-carboxylic acid isopropyl
ester
##STR00077##
[0256] The title compound was prepared from
3-((R)-1-carbamoylethoxy)pyrrolidine-1-carboxylic acid isopropyl
ester (Preparation 59) employing the procedure outlined in
Preparation 11: RT=3.06 min, m/z (ES.sup.+)=227.1 [M+H].sup.+.
Preparation 61
3-[(R)-1-(N-Hydroxycarbamimidoyl)ethoxy]pyrrolidine-1-carboxylic
acid isopropyl ester
##STR00078##
[0258] The title compound was prepared from
3-((R)-cyanomethylmethoxy)pyrrolidine-1-carboxylic acid isopropyl
ester (Preparation 60) employing the procedure outlined in
Preparation 12, but heating the reaction for 2 h: RT=1.99 min, m/z
(ES.sup.+)=260.1 [M+H].sup.+.
Preparation 62
3-{(R)-1-[5-(2-Chloropyrimidin-5-yl)-[1,2,4]oxadiazol-3-yl]ethoxy}-pyrroli-
dine-1-carboxylic acid isopropyl ester
##STR00079##
[0260] To a solution of 2-chloropyrimidin-2-ol (103 mg, 0.65 mmol)
in THF (10 mL), cooled to 0.degree. C., was added
1,3-diisopropylcarbodiimide (101 .mu.L, 0.65 mmol) and the reaction
was stirred for 10 min at this temperature.
3-[(R)-1-(N-Hydroxycarbamimidoyl)ethoxy]pyrrolidine-1-carboxylic
acid isopropyl ester (Preparation 61, 169 mg, 0.65 mmol) was added,
the ice bath was removed, and the mixture was allowed to stir at
r.t. for 1 h. The reaction solvent was concentrated to dryness,
then the resulting residue was dissolved in EtOAc, washed with
water, brine, then dried (Na.sub.2SO.sub.4), and the solvent
removed in vacuo. The residue was dissolved in toluene and heated
to 80.degree. C. for 18 h before being heated to reflux for 1 h.
Removal of the solvent in vacuo and purification by column
chromatography (IH:EtOAc, 1:1) afforded the title compound: RT=3.53
min, m/z (ES.sup.+)=382.1 [M+H].sup.+.
Preparation 63
1-Piperidin-4-yl ethanol
##STR00080##
[0262] To a solution of .alpha.-methyl-4-pyridine methanol (3.7 g,
30 mmol) in EtOH (100 mL) was added AcOH (1.9 mL, 33 mmol) and
platinum oxide (0.5 g, 2.2 mmol) and the resulting mixture was
allowed to stir under an atmosphere of hydrogen at r.t for 16 h.
The mixture was filtered and the filtrate was concentrated in
vacuo. The residue was dissolved in MeOH, to which was added a
solution of NaOH (1.6 g, 40 mmol) and water (1.6 mL) in MeOH. The
reaction was stirred for 30 min before removing the solvent in
vacuo, and the resulting residue was suspended in diethyl ether for
30 min. The mixture was filtered and the filtrate was concentrated
in vacuo to afford the title compound: .sup.1H NMR .delta..sub.H
(400 MHz, CDCl.sub.3): 3.63-3.55 (m, 1H), 3.39-3.31 (m, 2H),
2.7-2.6 (m, 2H), 2.01-1.92 (m, 2H), 1.76-1.69 (m, OH), 1.67-1.54
(m, 2H), 1.51-1.42 (m, 1H), 1.1-1.14 (m, 3H).
Preparation 64
4-(1-Hydroxyethyl)piperidine-1-carboxylic acid isopropyl ester
##STR00081##
[0264] To a solution 1-piperidin-4-yl ethanol (Preparation 63, 5.0
g, 38.76 mmol) in DCM (200 mL) in a 3-necked flask under argon, was
added DIPEA (8.8 mL, 50.39 mmol) and the reaction was cooled to
0.degree. C. A solution of isopropylchloroformate in toluene (46.5
mL, 46.5 mmol) was added dropwise, over 10 min, then the reaction
was brought to r.t. and stirred for a further 2.5 h. The reaction
mixture was diluted with DCM and partitioned with 1M HCl solution.
The organic layer was separated, washed with 1M HCl solution,
brine, and passed through a phase separator. Removal of the solvent
in vacuo afforded the title compound: .sup.1H NMR 3 (400 MHz,
CDCl.sub.3): 4.97-4.87 (m, 1H), 4.28-4.14 (m, 2H), 3.66-3.55 (m,
1H), 2.77-2.63 (m, 2H), 1.88-1.81 (m, 1H), 1.67-1.59 (m, 1H),
1.48-1.38 (m, 1H), 1.26-1.16 (m, 11H).
Preparation 65
4-[1-(5-Bromopyridin-2-yloxy)ethyl]piperidine-1-carboxylic acid
isopropyl ester
##STR00082##
[0266] A dry solution of 4-(1-hydroxyethyl)piperidine-1-carboxylic
acid isopropyl ester (Preparation 64, 7.4 g, 34.4 mmol) in DMF (100
mL), under argon, was cooled to 0.degree. C. Sodium hydride (60% in
mineral oil, 2.8 g, 68.8 mmol) was added in one portion, then the
reaction was allowed to stir at r.t. for 1 h.
5-bromo-2-chloropyridine (13.2 g, 68.8 mmol) was added and the
reaction was heated to 80.degree. C. for 16 h. The reaction mixture
was allowed to cool to r.t. and partitioned between EtOAc and
water. The organic phase was separated, washed with water, then
brine, and dried (MgSO.sub.4). Removal of the solvent in vacuo,
followed by trituration from iso-hexane (2.times.6 mL) then diethyl
ether, afforded the title compound: RT=4.34 min; ink
(ES.sup.+)=371.2 [M+H].sup.+.
Preparation 66
4-{1-[5-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)pyridin-2-yloxy]ethy-
l}piperidine-1-carboxylic acid isopropyl ester
##STR00083##
[0268] To a solution of
4-[1-(5-bromopyridin-2-yloxy)ethyl]piperidine-1-carboxylic acid
isopropyl ester (Preparation 65, 4.2 g, 11.3 mmol) in dioxane (90
mL) was added Potassium acetate (3.3 g, 33.9 mmol),
[1,1-bis(diphenylphosphino)ferrocene]dichloropalladium (0.9 g, 1.1
mmol) and bis(pinacolato)diboron (3.4 g, 13.4 mmol). The reaction
mixture was bubbled with argon for 10 min, before being heated to
110.degree. C. for 16 h. Removal of the solvent in vacuo followed
by purification of the crude material by column chromatography
(IH:EtOAc, 100:0, 97.5:2.5, 95:5) afforded the title compound:
RT=4.55 min; m/z (ES.sup.+)=419.4 [M+H].sup.+.
Preparation 67
(3S,4S)-4-Azido-1-benzylpyrrolidin-3-ylamine
##STR00084##
[0270] To a solution of (3S,4S)-3,4-diazido-1-benzylpyrrolidine
(15.6 g, 64.10 mmol) in THF (500 mL) cooled to 0.degree. C. was
added a solution of triphenylphosphine (16.5 g, 62.81 mmol) in THF
(100 mL), dropwise over 4 h and the resulting mixture was allowed
to reach r.t. and stirred for 16 h. The reaction solvent was
removed in vacuo and the resulting residue was re-dissolved in THF
(500 mL) and water (1.3 mL) before being heated to reflux for 4 h
then stirred at r.t. for 16 h. The reaction solvent was removed in
vacuo and the resulting residue was triturated with Et.sub.2O. The
precipitate was filtered and the filtrate was concentrated in
vacuo. The residue was taken into Et.sub.2O again and filtered.
Removal of the filtrate in vacuo followed by purification by column
chromatography (IH:EtOAc, 90:10, 80:20, 50:50, 0:100 then
MeOH:NH.sub.4OH, 9:1) afforded the title compound: RT=0.77 min; m/z
(ES.sup.+)=218.1 [M+H].sup.+.
Preparation 68
((3S,4S)-4-Azido-1-benzylpyrrolidin-3-yl)carbamic acid tert-butyl
ester
##STR00085##
[0272] To a solution of
(3S,4S)-4-azido-1-benzylpyrrolidin-3-ylamine (Preparation 67, 6.0
g, 27.74 mmol) and triethylamine (4.6 mL, 33.29 mmol) in DCM (100
mL), cooled to 0.degree. C., was added a solution of di
tert-butyldicarbonate (7.3 g, 33.29 mmol) in DCM (10 mL) dropwise
over 20 min. The resulting mixture was allowed to reach r.t. and
stirred for 72 h. The reaction solvent was washed with sat.
NaHCO.sub.3 solution, then brine, and dried (MgSO.sub.4). Removal
of the solvent in vacuo followed by purification by column
chromatography (DCM:MeOH) afforded the title compound: .sup.1H NMR
.delta..sub.H (400 MHz, CDCl.sub.3): 7.37-7.26 (m, 5H), 4.09-4.02
(m, 1H), 3.84-3.76 (m, 1H), 3.68-3.59 (m, 2H), 3.12-3.01 (m, 1H),
2.91-2.82 (m, 1H), 2.55-2.35 (m, 2H), 1.46 (s, 9H).
Preparation 69
((3S,4S)-4-Amino-1-benzylpyrrolidin-3-yl)carbamic acid tert-butyl
ester
##STR00086##
[0274] The title compound was prepared from
((3S,4S)-4-azido-1-benzylpyrrolidin-3-yl)carbamic acid tert-butyl
ester (Preparation 68) employing the procedure outlined in
WO2007/148185.
Preparation 70
[(3S,4S)-1-Benzyl-4-(2-oxopiperidin-1-yl)pyrrolidin-3-yl]carbamic
acid tert-butyl ester
##STR00087##
[0276] The title compound was prepared in 2 steps from
((3S,4S)-4-amino-1-benzylpyrrolidin-3-yl)carbamic acid tert-butyl
ester (Preparation 69) employing the procedures outlined in
WO2007/148185.
Preparation 71
[(3S,4S)-4-(2-Oxopiperidin-1-yl)pyrrolidin-3-yl]carbamic acid
tert-butyl ester
##STR00088##
[0278] A solution of
[(3S,4S)-1-benzyl-4-(2-oxopiperidin-1-yl)pyrrolidin-3-yl]carbamic
acid tert-butyl ester (Preparation 70, 2.6 g, 7.07 mmol) in MeOH
(140 mL) was passed through an H-Cube apparatus (10% pd/C Catcart
70, 10 bar, 90.degree. C.) at a flow rate of 1 mL per min. The
solvent was removed in vacuo to afford the title compound: .sup.1H
NMR .delta..sub.H (400 MHz, CDCl.sub.3): 5.25-5.07 (m, 1H),
4.85-4.62 (m, 1H), 4.34-4.07 (m, 1H), 3.49-3.28 (m, 3H), 3.24 (s,
1H), 2.99 (s, 1H), 2.87-2.73 (m, 1H), 2.52-2.39 (m, 2H), 2.38-2.22
(m, 2H), 1.91-1.74 (m, 1H), 1.54-1.38 (m, 9H).
Preparation 72
[(3S,4S)-1-(5-Bromopyrimidin-2-yl)-4-(2-oxopiperidin-1-yl)pyrrolidin-3-yl]-
carbamic acid tert-butyl ester
##STR00089##
[0280] A combination of
[(3S,4S)-4-(2-oxopiperidin-1-yl)pyrrolidin-3-yl]carbamic acid
tert-butyl ester (Preparation 71, 415 mg, 1.46 mmol),
5-bromo-2-chloropyrimidine (283 mg, 1.46 mmol) and DBU (219 .mu.L,
1.46 mmol) in DMSO (3 mL) was heated to 70.degree. C. for 1 h. The
reaction was allowed to cool to room temperature before being
partitioned between EtOAc and water. The organic phase was
separated, washed with brine, and dried (Na.sub.2SO.sub.4). Removal
of the solvent in vacuo, and purification by column chromatography
(IH:EtOAc, 1:1) afforded the title compound: RT=3.44 min, m/z
(ES.sup.+)=440.1 [M+H].sup.+.
Preparation 73
[(3R,4S)-1-(5-Bromopyrimidin-2-yl)-4-(2,5-difluorophenyl)pyrrolidin-3-yl]c-
arbamic acid tert-butyl ester
##STR00090##
[0282] A combination of
[(3R,4S)-4-(2,5-difluorophenyl)pyrrolidin-3-yl]carbamic acid
tert-butyl ester (Preparation 48, 540 mg, 1.8 mmol),
5-bromo-2-chloropyrimidine (350 mg, 1.8 mmol) and DBU (270 .mu.L,
1.8 mmol) in DMSO (3 mL) was heated to 70.degree. C. for 30 min.
The reaction was allowed to cool to room temperature before being
partitioned between EtOAc and water. The organic phase was
separated, washed with brine, and dried (Na.sub.2SO.sub.4). Removal
of the solvent in vacuo and purification by column chromatography
(IH:EtOAc, 1:1) afforded the title compound: RT=4.32 min, m/z
(ES.sup.+)=455.1 [M+H].sup.+.
Example 1
4-(5-{6-[(3R,4S)-3-Amino-4-(2-fluorophenyl)pyrrolidin-1-yl]pyridine-3-yl}--
[1,2,4]oxadiazol-3-ylmethoxy)piperidine-1-carboxylic acid isopropyl
ester
##STR00091##
[0284] A combination of
4-[5-(6-chloropyridin-3-yl)-[1,2,4]oxadiazol-3-ylmethoxy]piperidine-1-car-
boxylic acid isopropyl ester (Preparation 5, 150 mg, 0.39 mmol),
[(trans)-4-(2-fluorophenyl)pyrrolidin-3-yl]carbamic
acid-9H-fluoren-9-ylmethyl ester hydrochloride (Preparation 30),
172 mg, 0.39 mmol) and DIPEA (143 .mu.L, 0.82 mmol) in tert-butanol
(2 mL) was heated to 80.degree. C. until the reaction was complete.
The mixture was diluted with a small volume of DCM and purified by
column chromatography (DCM:MeOH, 96:4). Further purification by
chiral HPLC (MTBE:EtOH:BA 80:20:0.1, 11 mL/min, 285 nm, RT=35.0
min) afforded the title compound: RT=2.75 min; m/z (ES.sup.+)=525.2
[M+H].sup.+.
Example 2
4-(5-{2-[(3R,4S)-3-Amino-4-(2-fluorophenyl)pyrrolidin-1-yl]pyrimidin-5-yl}-
-[1,2,4]oxadiazol-3-ylmethoxy)piperidine-1-carboxylic acid
isopropyl ester p-toluenesulfonic acid salt
##STR00092##
[0286] A combination of
4-[5-(6-chloropyrimidin-5-yl)-[1,2,4]oxadiazol-3-ylmethoxy]piperidine-1-c-
arboxylic acid isopropyl ester (Preparation 6, 150 mg, 0.39 mmol),
[(trans)-4-(2-fluorophenyl)pyrrolidin-3-yl]carbamic
acid-9H-fluoren-9-ylmethyl ester hydrochloride (Preparation 30),
172 mg, 0.39 mmol) and DIPEA (143 .mu.L, 0.83 mmol) in tert-butanol
(2 mL) was heated to 80.degree. C. for 16 h. The reaction mixture
was concentrated in vacuo and re-dissolved in DCM (150 mL). The
organic mixture was washed with water (50 mL), brine (50 mL), then
dried (MgSO.sub.4), and the solvent was removed in vacuo.
Purification by column chromatography (DCM:MeOH, 96:4) followed by
chiral HPLC (MeOH:THF:CHCl.sub.3 55:25:20, 11 ml/min, 285 nm,
RT=6.8 min) afforded the intermediate product
4-(5-{2-[(3R,4S)-3-(9H-fluoren-9-ylmethoxycarbonylamino)-4-(2-fluoropheny-
l)pyrrolidin-1-yl]pyrimidin-5-yl}-[1,2,4]oxadiazol-3-ylmethoxy)piperidine--
1-carboxylic acid isopropyl ester: RT=4.89 min; m/z
(ES.sup.+)=748.3 [M+H].sup.+. To a solution of the product in DCM
(1 mL), cooled to 0.degree. C., was added a solution of piperidine
(0.5 mL) in DCM (1 mL), dropwise. The mixture was stirred at this
temperature for 2 h before removal of the solvent in vacuo and
purification by column chromatography (DCM:MeOH, 98:2, 92:8). A
solution of TsOH (1 eq.) in MeOH (2 mL) was added to the product
before removal of the solvent in vacuo to afford the title
compound: RT=2.78 min; m/z (ES.sup.+)=526.4 [M+H].sup.+.
Example 3
4-(5-{2-[(3R,4S)-3-Amino-4-(2,5-difluorophenyl)pyrrolidin-1-yl]pyrimidin-5-
-yl}-[1,2,4]oxadiazol-3-ylmethoxy)piperidine-1-carboxylic acid
isopropyl ester p-toluenesulfonic acid salt
##STR00093##
[0288] A combination of
4-[5-(6-chloropyrimidin-5-yl)-[1,2,4]oxadiazol-3-ylmethoxy]piperidine-1-c-
arboxylic acid isopropyl ester (Preparation 6, 65 mg, 0.17 mmol),
[(3R,4S)-4-(2,5-difluorophenyl)pyrrolidin-3-yl]carbamic acid
tert-butyl ester (Preparation 48, 59 mg, 0.17 mmol) and DIPEA (38
.mu.L, 0.22 mmol) in tert-butanol (1 mL) was heated to 80.degree.
C. until the reaction was complete. The reaction mixture was
concentrated in vacuo and purified by column chromatography
(DCM:MeOH, 99:1, 96:4) to afford the intermediate product
4-(5-{2-[(3R,4S)-3-tert-butoxycarbonylamino-4-(2,5-difluorophenyl-
)pyrrolidin-1-yl]pyrimidin-5-yl}-[1,2,4]oxadiazol-3-ylmethoxy)piperidine-1-
-carboxylic acid isopropyl ester: RT=4.47 min; m/z (ES.sup.+)=644.5
[M+H].sup.+. To a solution of the product in DCM (5 mL), cooled to
0.degree. C., was added TFA (1 mL), dropwise, and the reaction was
stirred at r.t. for 45 min. The mixture was diluted with DCM (75
mL), washed with sat. Na.sub.2CO.sub.3 solution (25 mL), then dried
(MgSO.sub.4), and the solvent was removed in vacuo. Purification by
column chromatography (DCM:MeOH, 96:4) afforded the title compound
as the free amine. A solution of TsOH (1 eq.) in MeOH (2 mL) was
added to the product and the solvent was removed in vacuo to afford
the title compound: RT=2.79 min; m/z (ES.sup.+)=544.2
[M+H].sup.+.
Example 4
4-(5-{2-[(3R,4S)-3-Amino-4-(2,4-difluorophenyl)pyrrolidin-1-yl]pyrimidin-5-
-yl}-[1,2,4]oxadiazol-3-ylmethoxy)piperidine-1-carboxylic acid
isopropyl ester p-toluenesulfonic acid salt
##STR00094##
[0290] The title compound was prepared from
4-[5-(6-chloropyrimidin-5-yl)-[1,2,4]oxadiazol-3-ylmethoxy]piperidine-1-c-
arboxylic acid isopropyl ester (Preparation 6) and
[(3R,4S)-4-(2,4-difluorophenyl)pyrrolidin-3-yl]carbamic acid
tert-butyl ester (Preparation 37) employing the procedure outlined
in Example 3: RT=2.75 min; m/z (ES.sup.+)=544.3 [M+H].sup.+.
Example 5
4-(3-{6-[(3R,4S)-3-Amino-4-(2-fluorophenyl)pyrrolidin-1-yl]pyridine-3-yl}--
[1,2,4]oxadiazol-5-ylmethoxy)piperidine-1-carboxylic acid isopropyl
ester p-toluenesulfonic acid salt
##STR00095##
[0292]
4-[3-(6-Chloropyridin-3-yl)-[1,2,4]oxadiazol-5-ylmethoxy]piperidine-
-1-carboxylic acid isopropyl ester (Preparation 7) was reacted with
[(trans)-4-(2-fluorophenyl)pyrrolidin-3-yl]carbamic
acid-9H-fluoren-9-ylmethyl ester hydrochloride (Preparation 30)
employing the procedure outlined in Example 1 to give the title
compound as the free amine (chiral HPLC: MTBE:MeOH:BA 80:20:0.1, 12
mL/min, 285 nm, RT=32.8 min) A solution of TsOH (1 eq.) in MeOH (5
mL) was added to the product and the solvent was removed in vacuo
to afford the title compound: RT=2.80 min; m/z (ES.sup.+)=525.2
[M+H].sup.+.
Example 6
4-(3-{6-[(3S,4R)-3-Amino-4-(2-fluorophenyl)pyrrolidin-1-yl]pyridine-3-yl}--
[1,2,4]oxadiazol-5-ylmethoxy)piperidine-1-carboxylic acid isopropyl
ester p-toluenesulfonic acid salt
##STR00096##
[0294] The title compound was prepared from
4-[3-(6-chloropyridin-3-yl)-[1,2,4]oxadiazol-5-ylmethoxy]piperidine-1-car-
boxylic acid isopropyl ester (Preparation 7) and
[(trans)-4-(2-fluorophenyl)pyrrolidin-3-yl]carbamic
acid-9H-fluoren-9-ylmethyl ester hydrochloride (Preparation 30),
employing the procedure outlined in Example 5. Chiral HPLC:
MTBE:MeOH:BA 80:20:0.1, 12 mL/min, 285 nm, RT=29.2 min LCMS:
RT=2.80 min, m/z (ES.sup.+)=525.2 [M+H].sup.+.
Example 7
4-(3-{6-[(3S,4R)-3-Amino-4-(2-fluorophenyl)pyrrolidin-1-yl]pyridine-3-yl}--
[1,2,4]oxadiazol-5-ylmethoxy)piperidine-1-carboxylic acid isopropyl
ester p-toluenesulfonic acid salt
##STR00097##
[0296] The title compound was prepared from
4-{(R-1-[3-(6-chloropyridin-3-yl)-[1,2,4]oxadiazol-5-yl]ethoxy}piperidine-
-1-carboxylic acid isopropyl ester (Preparation 8) and
[(trans)-4-(2-fluorophenyl)pyrrolidin-3-yl]carbamic
acid-9H-fluoren-9-ylmethyl ester hydrochloride (Preparation 30),
employing the procedure outlined in Example 5. Chiral HPLC:
MTBE:MeOH:BA 80:20:0.1, 12 mL/min, 285 nm RT=20.4 min. LCMS:
RT=2.89 min; m/z (ES.sup.+)=539.5 [M+H].sup.+.
Example 8
4-(3-{6-[(3R,4S)-3-Amino-4-(2,5-difluorophenyl)pyrrolidin-1-yl]pyridin-3-y-
l}-[1,2,4]oxadiazol-5-ylmethoxy)piperidine-1-carboxylic acid
isopropyl ester p-toluenesulfonic acid salt
##STR00098##
[0298]
4-[3-(6-Chloropyridin-3-yl)-[1,2,4]oxadiazol-5-ylmethoxy]piperidine-
-1-carboxylic acid isopropyl ester (Preparation 7) was reacted with
[(3R,4S)-4-(2,5-difluorophenyl)pyrrolidin-3-yl]carbamic acid
tert-butyl ester (Preparation 48) employing the procedure outlined
in Example 3. After reaction with TFA the crude mixture was passed
down an SCX cartridge, eluting with MeOH then NH.sub.4OH in MeOH,
and the basic fraction was collected to afford the title compound
as the free amine. To a solution of the product in MeOH was added
TsOH (1 eq.) in MeOH, then the solvent was removed in vacuo to
afford the title compound: RT=2.82 min; m/z (ES.sup.+)=543.2
[M+H].sup.+.
Example 9
4-[(R)-1-(3-{6-[(3R,4S)-3-Amino-4-(2,5-difluorophenyl)pyrrolidin-1-yl]pyri-
din-3-yl}-[1,2,4]oxadiazol-5-yl)ethoxy]piperidine-1-carboxylic acid
isopropyl ester p-toluenesulfonic acid salt
##STR00099##
[0300] A combination of
4-{(R-1-[3-(6-chloropyridin-3-yl)-[1,2,4]oxadiazol-5-yl]ethoxy}piperidine-
-1-carboxylic acid isopropyl ester (Preparation 8, 79 mg, 0.20
mmol), [(3R,4S)-4-(2,5-difluorophenyl)pyrrolidin-3-yl]carbamic acid
tert-butyl ester (Preparation 48, 60 mg, 0.20 mmol) and DIPEA (38
.mu.L, 0.22 mmol) in tert-butanol (1 mL) was heated to 80.degree.
C. for 72 h and then 85.degree. C. until no further reaction. The
mixture was concentrated in vacuo and purified by column
chromatography (DCM:MeOH, 99:1, 97:3) to afford the intermediate
product
4-[(R)-1-(3-{6-[(3R,4S)-3-tert-butoxycarbonylamino-4-(2,5-difluorophenyl)-
pyrrolidin-1-yl]pyridin-3-yl}-[1,2,4]oxadiazol-5-yl)ethoxy]piperidine-1-ca-
rboxylic acid isopropyl ester: RT=4.39 min; m/z (ES.sup.+)=657.4
[M+H].sup.+. To a solution of the product in DCM (5 mL), cooled to
0.degree. C., was added TFA (1 mL), dropwise, and the reaction was
stirred at r.t. for 45 min. The mixture was diluted with DCM,
washed with sat. Na.sub.2CO.sub.3 solution, dried (MgSO.sub.4), and
the solvent removed in vacuo. Purification by column chromatography
(DCM:MeOH, 97:3) afforded the title compound as the free amine. A
solution of TsOH (1 eq.) in MeOH (2 mL) was added to the product,
then the solvent was removed in vacuo to afford the title compound:
RT=2.93 min; m/z (ES.sup.+)=557.3 [M+H].sup.+.
Example 10
4-[(R)-1-(5-{2-[(3R,4S)-3-Amino-4-(2,5-difluorophenyl)pyrrolidin-1-yl]pyri-
midin-5-yl}-[1,2,4]oxadiazol-3-yl)ethoxy]piperidine-1-carboxylic
acid isopropyl ester
##STR00100##
[0302]
4-{(R)-1-[5-(2-Chloropyrimidin-5-yl)-[1,2,4]oxadiazol-3-yl]ethoxy}p-
iperidine-1-carboxylic acid isopropyl ester (Preparation 13) was
reacted with
[(3R,4S)-4-(2,5-difluorophenyl)pyrrolidin-3-yl]carbamic acid
tert-butyl ester (Preparation 48) employing the procedure outlined
in Example 3. After reaction with TFA the crude mixture was passed
down an SCX cartridge, eluting with MeOH then NH.sub.4OH in MeOH,
and the basic fraction was collected to afford the title compound:
RT=2.80 min; m/z (ES.sup.+)=558.2 [M+H].sup.+.
Example 11
4-[(R)-1-(5-{5-[(3R,4S)-3-Amino-4-(2,5-difluorophenyl)pyrrolidin-1-yl]pyra-
zin-2-yl}-[1,2,4]oxadiazol-3-yl)ethoxy]piperidine-1-carboxylic acid
isopropyl ester
##STR00101##
[0304] To a solution of
4-{(R)-1-[5-(5-chloropyrazin-2-yl)-[1,2,4]oxadiazol-3-yl]ethoxy}piperidin-
e-1-carboxylic acid isopropyl ester (Preparation 14, 127 mg, 0.32
mmol) in DMSO (0.5 mL) was added
[(3R,4S)-4-(2,5-difluorophenyl)pyrrolidin-3-yl]carbamic acid
tert-butyl ester (Preparation 48, 98 mg, 0.33 mmol) and DBU (48
.mu.L, 0.32 mmol), and the reaction was heated to 70.degree. C. for
1 h. The mixture was partitioned between DCM (100 mL) and water (50
mL), and the organic phase was separated. The aqueous phase was
extracted with DCM (50 mL), then the organic fractions were
combined, washed with brine, and dried (MgSO.sub.4). Purification
by column chromatography (DCM:MeOH, 98:2) afforded the intermediate
product
4-[(R)-1-(5-{5-[(3R,4S)-3-tert-butoxycarbonylamino-4-(2,5-difluorophenyl)-
pyrrolidin-1-yl]pyrazin-2-yl}-[1,2,4]oxadiazol-3-yl)ethoxy]piperidine-1-ca-
rboxylic acid isopropyl ester: RT=4.43 min; m/z (ES.sup.+)=658.3
[M+H].sup.+. To a solution of the product in DCM (5 mL), cooled to
0.degree. C., was added TFA (1 mL), dropwise, and the reaction was
stirred at r.t. until complete. Removal of the solvent in vacuo and
purification by column chromatography (DCM:MeOH, 95:5) afforded the
title compound: RT=2.74 min; m/z (ES.sup.+)=558.2 [M+H].sup.+.
Example 12
4-{5-[(3R,4R)-3-Amino-4-(2,5-difluorophenyl)-3,4,5,6-tetrahydro-2H-[1,2]bi-
pyridinyl-5'yl]-[1,2,4]oxadiazol-3-ylmethoxy}piperidine-1-carboxylic
acid isopropyl ester hydrochloride
##STR00102##
[0306] A combination of
4-[5-(6-chloropyridin-3-yl)-[1,2,4]oxadiazol-3-ylmethoxy]piperidine-1-car-
boxylic acid isopropyl ester (Preparation 5, 38 mg, 0.10 mmol),
[(3R,4R)-4-(2,5-difluorophenyl)piperidin-3-yl]carbamic acid
tert-butyl ester (Preparation 19, 31 mg, 0.10 mmol), and DIPEA (18
.mu.L, 0.11 mmol) in tert-butanol (0.30 mL) was heated to
80.degree. C. until the reaction was complete. The mixture was
diluted with EtOAc (50 mL), washed with brine (50 mL), dried
(MgSO.sub.4), and concentrated in vacuo to afford the intermediate
product
4-{5-[(3R,4R)-3-tert-butylcarbonylamino-4-(2,5-difluorophenyl)-3,4,5,6-te-
trahydro-2H-[1,2']bipyridinyl-5'yl]-[1,2,4]oxadiazol-3-ylmethoxy}piperidin-
e-1-carboxylic acid isopropyl ester: RT=4.53 min; m/z
(ES.sup.+)=657.2 [M+H].sup.+. To a solution of the product in DCM
(3 mL) was added TFA (3 mL) and the reaction was stirred at r.t.
for 30 min. The crude mixture was passed down an SCX cartridge,
eluting with MeOH then NH.sub.4OH in MeOH, and the basic fraction
was collected and concentrated in vacuo to afford the title
compound as the free amine. The residue was dissolved in a solution
of HCl in dioxane (4M), then the solvent was removed in vacuo to
afford the title compound: RT=2.85 min; m/z (ES.sup.+)=557.3
[M+H].sup.+.
Example 13
4-{3-[(3R,4R)-3-Amino-4-(2,5-difluorophenyl)-3,4,5,6-tetrahydro-2H-[1,2]bi-
pyridinyl-5'-yl]-[1,2,4]oxadiazol-5-ylmethoxy}piperidine-1-carboxylic
acid isopropyl ester dihydrochloride
##STR00103##
[0308] The title compound was prepared from
4-[3-(6-chloropyridin-3-yl)-[1,2,4]oxadiazol-5-ylmethoxy]piperidine-1-car-
boxylic acid isopropyl ester (Preparation 7) and
[(3R,4R)-4-(2,5-difluorophenyl)piperidin-3-yl]carbamic acid
tert-butyl ester (Preparation 19) employing the procedure outlined
in Example 12. After purification by SCX cartridge the residue was
further purified by preparative HPLC. Salt formation employing the
method outlined in Example 12 afforded the title compound as the
dihydrochloride salt: RT=2.73 min; m/z (ES.sup.+)=557.2
[M+H].sup.+.
Example 14
4-(3-{2-[(3R,4S)-3-Amino-4-(2,4-difluorophenyl)pyrrolidin-1-yl]pyrimidin-5-
-yl}-[1,2,4]oxadiazol-5-ylmethoxy)piperidine-1-carboxylic acid
isopropyl ester p-toluenesulfonic acid salt
##STR00104##
[0310] To a solution of
4-[3-(2-chloropyrimidin-5-yl)-[1,2,4]oxadiazol-5-ylmethoxy]piperidine-1-c-
arboxylic acid isopropyl ester (Preparation 25, 40 mg, 0.10 mmol)
and [(3R,4S)-4-(2,4-difluorophenyl)pyrrolidin-3-yl]carbamic acid
tert-butyl ester (Preparation 37, 38 mg, 0.13 mmol) in DMSO (0.4
mL) was added DBU (16 .mu.L, 0.10 mmol), and the mixture was
bubbled with argon for 1 min before being heated to 70.degree. C.
for 16 h. The reaction mixture was partitioned between EtOAc (50
mL) and water (20 mL), and the organic phase was separated. The
aqueous phase was extracted with EtOAc (20 mL) then organic
fractions were combined, washed with sat. NaHCO.sub.3 solution (50
mL), brine (50 mL), then dried (MgSO.sub.4), and the solvent was
removed in vacuo. Purification by column chromatography (DCM:MeOH,
100:0, 98:2) afforded the intermediate product
4-(3-{2-[(3R,4S)-3-tert-butoxycarbonylamino-4-(2,4-difluorophenyl)pyrroli-
din-1-yl]pyrimidin-5-yl}-[1,2,4]oxadiazol-5-ylmethoxy)piperidine-1-carboxy-
lic acid isopropyl ester: RT=4.43 min; m/z (ES.sup.+)=644.3
[M+H].sup.+. To a solution of the product in DCM (5 mL), under
argon, cooled to 0.degree. C., was added TFA (1 mL) and the
reaction was stirred at this temperature for 2 h. Further TFA (0.5
mL) was added and stirring continued until the reaction was
complete. The crude mixture was passed down an SCX cartridge,
eluting with MeOH then NH.sub.4OH in MeOH. The basic fraction was
collected and concentrated in vacuo to afford the title compound as
the free amine. To a solution of the product in DCM (2 mL) was
added a solution of TsOH (1 eq.) in MeOH (2 mL), then the solvent
was removed in vacuo to afford the title compound: RT=2.86 min; m/z
(ES.sup.+)=544.2 [M+H].sup.+.
[0311] The following examples were prepared by reacting the
appropriate 2-chloropyrimidine intermediate with the appropriate
amine building block employing the procedure employed in Example
14:
TABLE-US-00003 Ex. Structure Name LCMS 15 ##STR00105##
4-(3-{2-[(3R,4S)-3- Amino-4-(2,5- difluorophenyl)pyrrolidin-
1-yl]pyrimidin-5-yl}- [1,2,4]oxadiazol-5- ylmethoxy)piperidine-1-
carboxylic acid isopropyl ester p-toluenesulfonic acid salt RT =
2.82 min, m/z (ES.sup.+) = 544.2 [M + H].sup.+ 16 ##STR00106##
4-(3-{2-[(3R,4R)-3- Amino-4-(2,5- difluorophenyl)piperidin-
1-yl]pyrimidin-5-yl}- [1,2,4]oxadiazol-5- ylmethoxy)piperidine-1-
carboxylic acid isopropyl ester p-toluenesulfonic acid salt RT =
2.92 min, m/z (ES.sup.+) = 558.2 [M + H].sup.+ 17 ##STR00107##
4-[(R)-1-(3-{2-[(3R,4S)- 3-Amino-4-(2,4- difluorophenyl)pyrrolidin-
1-yl]pyrimidin-5-yl}- [1,2,4]oxadiazol-5- yl)ethoxy]piperidine-1-
carboxylic acid isopropyl ester p-toluenesulfonic acid salt RT =
2.93 min, m/z (ES.sup.+) = 558.2 [M + H].sup.+ 18 ##STR00108##
4-[(R)-1-(3-{2-[(3R,4S)- 3-Amino-4-(2,5- difluorophenyl)pyrrolidin-
1-yl]pyrimidin-5-yl}- [1,2,4]oxadiazol-5- yl)ethoxy]piperidine-1-
carboxylic acid isopropyl ester p-toluenesulfonic acid salt RT =
2.94 min, m/z (ES.sup.+) = 558.2 [M + H].sup.+
Example 19
4-(3-{2-[(3R,4S)-3-Amino-4-(2-fluorophenyl)pyrrolidin-1-yl]pyrimidin-5-yl}-
-[1,2,4]oxadiazol-5-ylmethoxy)piperidine-1-carboxylic acid
isopropyl ester p-toluenesulfonic acid salt
##STR00109##
[0313] To a solution of
4-[3-(2-chloropyrimidin-5-yl)-[1,2,4]oxadiazol-5-ylmethoxy]piperidine-1-c-
arboxylic acid isopropyl ester (Preparation 25, 19 mg, 0.05 mmol)
and [(3R,4S)-4-(2-fluorophenyl)pyrrolidin-3-yl]carbamic
acid-9H-fluoren-9-ylmethyl ester hydrochloride (Preparation 32, 31
mg, 0.07 mmol) in DMSO (0.4 mL), under argon, was added DBU (15
.mu.L, 0.10 mmol) and the reaction was heated to 80.degree. C. for
65 h. The mixture was diluted with water (10 mL) and the resulting
solution extracted with EtOAc (3.times.30 mL). The organic
fractions were combined, washed with water (30 mL), sat.
NaHCO.sub.3 solution (30 mL), and brine (30 mL), then dried
(MgSO.sub.4). Removal of the solvent in vacuo and purification by
column chromatography (DCM:MeOH, 100:0, 98:2, 97:3, 95:5, 92:8)
afforded the title compound as the free amine. To a solution of the
product in DCM (4 mL) was added a solution of TsOH (1 eq.) in MeOH
(2 mL), then the solvent was removed in vacuo to afford the title
compound: RT=2.74 min; m/z (ES.sup.+)=526.2 [M+H].sup.+.
Example 20
4-[(R)-1-(3-{2-[(3R,4S)-3-Amino-4-(2-fluorophenyl)pyrrolidin-1-yl]pyrimidi-
n-5-yl]-[1,2,4]oxadiazol-5-yl)ethoxy}piperidine-1-carboxylic acid
isopropyl ester p-toluenesulfonic acid salt
##STR00110##
[0315] The title compound was prepared by reacting
4-{(R)-1-[3-(2-chloropyrimidin-5-yl)-[1,2,4]oxadiazol-5-yl]ethoxy}piperid-
ine-1-carboxylic acid isopropyl ester (Preparation 28) with
[(3R,4S)-4-(2-fluorophenyl)pyrrolidin-3-yl]carbamic
acid-9H-fluoren-9-ylmethyl ester hydrochloride (Preparation 32)
employing the procedure outlined in Example 19: RT=2.84 min; m/z
(ES.sup.+)=540.3 [M+H].sup.+.
Example 21
4-[(R)-1-(5-{6-[(3R,4S)-3-Amino-4-(2,5-difluorophenyl)pyrrolidin-1-yl]pyri-
dazin-3-yl}-[1,2,4]oxadiazol-3-yl)ethoxy]piperidine-1-carboxylic
acid isopropyl ester
##STR00111##
[0317] A combination of
4-((R)-1-{5-[6-(benzotriazol-1-yloxy)pyridazin-3-yl]-[1,2,4]oxadiazol-3-y-
l}ethoxy)piperidine-1-carboxylic acid isopropyl ester (Preparation
38, 12 mg, 0.02 mmol) and
[(3R,4S)-4-(2,5-difluorophenyl)pyrrolidin-3-yl]carbamic acid
tert-butyl ester (Preparation 48, 8 mg, 0.03 mmol) in DMSO (0.5 mL)
was heated to 70.degree. C. for 1 h. The reaction mixture was
diluted with water and extracted with EtOAc (2.times.50 mL). The
organic fractions were combined, washed with brine, dried
(MgSO.sub.4) and the solvent removed in vacuo to afford the
intermediate product
4-[(R)-1-(5-{6-[(3R,4S)-3-tert-butoxycarbonylamino-4-(2,5-difluorophenyl)-
pyrrolidin-1-yl]pyridazin-3-yl}-[1,2,4]oxadiazol-3-yl)ethoxy]piperidine-1--
carboxylic acid isopropyl ester: RT=4.23 min; m/z (ES.sup.+)=658.3
[M+H].sup.+. To a solution of the product in DCM (5 mL), cooled to
0.degree. C., was added TFA (0.5 mL), dropwise, and the reaction
was stirred at r.t. for 1 h. The crude mixture was passed down an
SCX cartridge, eluting with MeOH then NH.sub.4OH in MeOH, and the
basic fraction was collected and concentrated in vacuo. Further
purification by column chromatography (DCM:MeOH, 95:5) afforded the
title compound: RT=2.72 min; m/z (ES.sup.+)=558.2 [M+H].sup.+.
Example 22
3-[(R)-1-(3-{2-[(3R,4S)-3-Amino-4-(2,5-difluorophenyl)pyrrolidin-1-yl]pyri-
midin-5-yl}-[1,2,4]oxadiazol-5-yl)ethoxy]azetidine-1-carboxylic
acid isopropyl ester p-toluenesulfonic acid salt
##STR00112##
[0319] To a solution of
3-[(R)-1-(3-{2-[(3R,4S)-3-tert-butoxycarbonylamino-4-(2,5-difluorophenyl)-
pyrrolidin-1-yl]pyrimidin-5-yl}-[1,2,4]oxadiazol-5-yl)ethoxy]azetidine-1-c-
arboxylic acid isopropyl ester (Preparation 51,109 mg, 0.17 mmol)
in DCM (5 mL), cooled to 0.degree. C., was added TFA (1.0 mL),
dropwise, and the reaction was stirred at this temperature for 2 h.
A further portion of TFA (0.5 mL) was added and stirring continued
for 30 min. The crude reaction mixture was passed down an SCX
cartridge, eluting with MeOH then NH.sub.4OH in MeOH, and the basic
fraction was collected and concentrated in vacuo. To a solution of
the product in DCM (2 mL) was added a solution of TsOH (1 eq.) in
MeOH (2 mL), then the solvent was removed in vacuo to afford the
title compound: RT=2.73 min; m/z (ES.sup.+)=530.2 [M+H].sup.+.
Example 23
4-[(R)-1-(3-{5-[(3R,4S)-3-Amino-4-(2,5-difluorophenyl)pyrrolidin-1-yl]pyra-
zin-2-yl}-[1,2,4]oxadiazol-5-yl)ethoxy]piperidine-1-carboxylic acid
isopropyl ester
##STR00113##
[0321] To a solution of
4-((R)-1-carboxyethoxy)piperidine-1-carboxylic acid isopropyl ester
(Preparation 9, 72 mg, 0.28 mmol) in THF (20 mL) was added EDCI (63
mg, 0.33 mmol), followed by HOBt (45 mg, 0.30 mmol), and the
reaction was stirred at r.t. for 10 min.
{(3R,4S)-4-(2,5-Difluorophenyl)-1-[5-(N-hydroxycarbamimidoyl)pyrazin-2-yl-
]pyrrolidin-3-yl}carbamic acid tert-butyl ester (Preparation 53,
120 mg, 0.28 mmol) was added and the reaction was stirred at r.t.
for 16 h. The reaction solvent was concentrated in vacuo and the
resulting residue was partitioned between EtOAc (100 mL) and water
(50 mL). The organic phase was separated, dried (MgSO.sub.4), and
concentrated in vacuo. The residue was taken into toluene and
heated to reflux for 16 h. Removal of the solvent in vacuo and
purification by column chromatography (DCM:MeOH, 97:3) afforded the
intermediate product: RT=4.39 min; m/z (ES.sup.+)=658.3
[M+H].sup.+. To a solution of the product in DCM (7 mL), cooled to
0.degree. C., was added TFA (0.7 mL). The ice bath was removed and
the reaction was stirred at r.t. for 1 h. Purification by
preparative HPLC followed by column chromatography (DCM:MeOH, 95:5)
afforded the title compound: RT=2.79 min; m/z (ES.sup.+)=5581
[M+H].sup.+.
Example 24
4-[(R)-1-(5-{2-[(3R,4S)-3-Amino-4-(2,4,5-trifluorophenyl)pyrrolidin-1-yl]p-
yrimidin-5-yl}-[1,2,4]oxadiazol-3-yl)ethoxy]piperidine-1-carboxylic
acid isopropyl ester
##STR00114##
[0323] A combination of
4-{(R)-1-[5-(2-chloropyrimidin-5-yl)-[1,2,4]oxadiazol-3-yl]ethoxy}piperid-
ine-1-carboxylic acid isopropyl ester (Preparation 13, 100 mg, 0.25
mmol) and
[(3R,4S)-4-(2,4,5-trifluorophenyl)pyrrolidin-3-yl]carbamic acid
tert-butyl ester (Preparation 56, 80 mg, 0.25 mmol) in DMSO (1.0
mL) was treated with DBU (38 .mu.L, 0.25 mmol) and the reaction was
heated to 70.degree. C. for 3 h. The reaction mixture was diluted
with water (70 mL) and extracted with EtOAc (2.times.100 mL). The
organic fractions were combined, washed with brine, dried
(MgSO.sub.4) and the solvent was removed in vacuo to afford the
intermediate product
4-[(R)-1-(5-{2-[(3R,4S)-3-tert-butoxycarbonylamino-4-(2,4,5-trifluorophen-
yl)pyrrolidin-1-yl]pyrimidin-5-yl}-[1,2,4]oxadiazol-3-yl)ethoxy]piperidine-
-1-carboxylic acid isopropyl ester: RT=4.69 min; m/z
(ES.sup.+)=676.3 [M+H].sup.+. To a solution of the product in DCM
(7 mL), cooled to 0.degree. C., was added TFA (0.7 mL), dropwise,
and the resulting reaction was stirred at r.t. for 1 h. The crude
mixture was passed down an SCX cartridge, eluting with MeOH then
NH.sub.4OH in MeOH, and the basic fraction was collected and
concentrated in vacuo to afford the title compound: RT=2.88 min;
m/z (ES.sup.+)=576.2 [M+H].sup.+.
Example 25
(R)-3-[(R)-1-(5-{2-[(3R,4S)-3-Amino-4-(2,5-difluorophenyl)pyrrolidin-1-yl]-
pyrimidin-5-yl}-[1,2,4]oxadiazol-3-yl)ethoxy]pyrrolidine-1-carboxylic
acid isopropyl ester
##STR00115##
[0325] A combination of
3-{(R)-1-[5-(2-chloropyrimidin-5-yl)-[1,2,4]oxadiazol-3-yl]ethoxy}pyrroli-
dine-1-carboxylic acid isopropyl ester (Preparation 62, 60 mg, 0.16
mmol), [(3R,4S)-4-(2,5-difluorophenyl)pyrrolidin-3-yl]carbamic acid
tert-butyl ester (Preparation 48, 48 mg, 0.16 mmol) and DIPEA (29
.mu.L, 0.18 mmol) in tert-butanol (1 mL) was heated to 80.degree.
C. until the reaction was complete The solvent was concentrated in
vacuo, then purification by column chromatography (IH:EtOAc, 1:1)
followed by chiral HPLC (IH:EtOH:THF 70:20:10, 15 ml/min, 290 nm,
RT=11.4 min) afforded the intermediate product
(R)-3-[(R)-1-(5-{2-[(3R,4S)-3-tert-butoxycarbonylamino-4-(2,5-difluorophe-
nyl)pyrrolidin-1-yl]pyrimidin-5-yl}-[1,2,4]oxadiazol-3-yl)ethoxy]pyrrolidi-
ne-1-carboxylic acid isopropyl ester: RT=4.49 min; m/z
(ES.sup.+)=644.5 [M+H].sup.+. To a solution of the product in DCM
(0.5 mL), cooled to 0.degree. C., was added TFA (0.05 mL) and the
reaction was stiffed for 2 h. A further portion of TFA (0.05 mL)
was added and stirring continued until the reaction was complete.
The crude mixture was passed down an SCX cartridge, eluting with
MeOH then NH.sub.4OH in MeOH (10%). The basic fraction was
collected and concentrated in vacuo to afford the title compound:
RT=2.83 min; ink (ES.sup.+)=544.0 [M+H].sup.+.
Example 26
(S)-3-[(R)-1-(5-{2-[(3R,4S)-3-Amino-4-(2,5-difluorophenyl)pyrrolidin-1-yl]-
pyrimidin-5-yl}-[1,2,4]oxadiazol-3-yl)ethoxy]pyrrolidine-1-carboxylic
acid isopropyl ester
##STR00116##
[0327] The title compound was prepared by reacting
3-{(R)-1-[5-(2-chloropyrimidin-5-yl)-[1,2,4]oxadiazol-3-yl]ethoxy}pyrroli-
dine-1-carboxylic acid isopropyl ester (Preparation 62) with
[(3R,4S)-4-(2,5-difluorophenyl)pyrrolidin-3-yl]carbamic acid
tert-butyl ester (Preparation 48) employing the procedure outlined
in Example 25. Chiral HPLC: IH:EtOH:THF 70:20:10, mL/min, 290 nm,
RT: 9.8 min. LCMS: RT=2.72 min; m/z (ES.sup.+)=544.2
[M+H].sup.+.
Example 27
4-[(S)-1-(5-{2-[(3S,4S)-3-Amino-4-(2-oxopiperidin-1-yl)pyrrolidin-1-yl]pyr-
imidin-5-yl}pyridin-2-yloxy)ethyl]piperidine-1-carboxylic acid
isopropyl ester
##STR00117##
[0329] To a solution of
4-{1-[5-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)pyridin-2-yloxy]eth-
yl}piperidine-1-carboxylic acid isopropyl ester (Preparation 66,
350 mg, 0.84 mmol) and
[(3S,4S)-1-(5-bromopyrimidin-2-yl)-4-(2-oxopiperidin-1-yl)pyrrolidin-3-yl-
]carbamic acid tert-butyl ester (Preparation 72, 442 mg, 1.00 mmol)
in a combination of DMF (7 mL) and water (1.8 mL) was added
[1,1-bis(diphenylphosphino)ferrocene]dichloropalladium (82 mg, 0.10
mmol) and triethylamine (418 mL, 3.00 mmol). The reaction was
heated in a microwave reactor at 80.degree. C. for 20 min, and then
reacted for a further 5 min at 80.degree. C., before being filtered
through celite, washing with EtOAc. The organic mixture was washed
with water, 1M citric acid, sat. NaHCO.sub.3 solution, then brine,
and dried (Na.sub.2SO.sub.4), before removal of the solvent in
vacuo. Purification by column chromatography (IH:EtOAc, 1:1,
0:100), followed by chiral HPLC (MeCN:THF:MeOH, 67:30:3, 1 ml/min,
285 nm, RT=7.48 min), afforded the intermediate product
4-[(S)-1-(5-{2-[(3S,4S)-3-tert-butoxycarbonylamino-4-(2-oxopiperidin-1-yl-
)pyrrolidin-1-yl]pyrimidin-5-yl}pyridin-2-yloxy)ethyl]piperidine-1-carboxy-
lic acid isopropyl ester: RT=4.18 min, m/z (ES.sup.+)=652.2
[M+H].sup.+. To a solution of the product in DCM (1 mL) was added
TFA (0.5 mL) and the reaction was stirred at r.t. for 1 h. The
crude mixture was passed directly down an SCX cartridge, eluting
with MeOH then NH.sub.4OH in MeOH (10%). The basic fraction was
collected and concentrated in vacuo to afford the title compound:
RT=2.83 min, m/z (ES.sup.+)=552.4 [M+H].sup.+.
Example 28
4-[(S)-1-(5-{2-[(3R,4S)-3-Amino-4-(2,5-difluorophenyl)pyrrolidin-1-yl]pyri-
midin-5-yl}pyridin-2-yloxy)ethyl]piperidine-1-carboxylic acid
isopropyl ester
##STR00118##
[0331] The title compound was prepared by reacting
4-{1-[5-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)pyridin-2-yloxy]eth-
yl}piperidine-1-carboxylic acid isopropyl ester (Preparation 66)
with
[(3R,4S)-1-(5-bromopyrimidin-2-yl)-4-(2,5-difluorophenyl)pyrrolidin-3-yl]-
carbamic acid tert-butyl ester (Preparation 73) employing the
procedure outlined in Example 27. Chiral HPLC conditions and RT of
intermediate MTBE:THF 85:15, 1 mL/min, 285 nm, RT=8.6 min. LCMS:
RT=3.00 min, m/z (ES.sup.+)=567.3 [M+H].sup.+.
Example 29
4-[(R)-1-(5-{2-[(3R,4S)-3-Amino-4-(2,5-difluorophenyl)pyrrolidin-1-yl]pyri-
midin-5-yl]pyridin-2-yloxy)ethyl}piperidine-1-carboxylic acid
isopropyl ester
##STR00119##
[0333] The title compound was prepared by reacting
4-{1-[5-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)pyridin-2-yloxy]eth-
yl}piperidine-1-carboxylic acid isopropyl ester (Preparation 66)
with
[(3R,4S)-1-(5-bromopyrimidin-2-yl)-4-(2,5-difluorophenyl)pyrrolidin-3-yl]-
carbamic acid tert-butyl ester (Preparation 73) employing the
procedure outlined in Example 27. Chiral HPLC conditions and RT of
intermediate MTBE:THF 85:15, 1 mL/min, 285 nm, RT=7.2 min. LCMS:
RT=3.10 min, m/z (ES.sup.+)=567.3 [M+H].sup.+.
[0334] The biological activity of the compounds of the invention
may be tested in the following assay systems:
GPR119 Yeast Reporter Assay
Yeast Reporter Assay
[0335] The yeast cell-based reporter assays have previously been
described in the literature (e.g. see Miret J. J. et al, 2002, J.
Biol. Chem., 277:6881-6887; Campbell R. M. et al, 1999, Bioorg.
Med. Chem. Lett., 9:2413-2418; King K. et al, 1990, Science,
250:121-123); WO 99/14344; WO 00/12704; and U.S. Pat. No.
6,100,042). Briefly, yeast cells have been engineered such that the
endogenous yeast G-alpha (GPA1) has been deleted and replaced with
G-protein chimeras constructed using multiple techniques.
Additionally, the endogenous yeast GPCR, Step 3 has been deleted to
allow for heterologous expression of a mammalian GPCR of choice. In
the yeast, elements of the pheromone signaling transduction
pathway, which are conserved in eukaryotic cells (for example, the
mitogen-activated protein kinase pathway), drive the expression of
Fus1. By placing .beta.-galactosidase (LacZ) under the control of
the Fus1 promoter (Fus1p), a system has been developed whereby
receptor activation leads to an enzymatic read-out.
[0336] Yeast cells were transformed by an adaptation of the lithium
acetate method described by Agatep et al, (Agatep, R. et al, 1998,
Transformation of Saccharomyces cerevisiae by the lithium
acetate/single-stranded carrier DNA/polyethylene glycol
(LiAc/ss-DNA/PEG) protocol. Technical Tips Online, Trends Journals,
Elsevier). Briefly, yeast cells were grown overnight on yeast
tryptone plates (YT). Carrier single-stranded DNA (10 .mu.g), 2
.mu.g of each of two Fus1p-LacZ reporter plasmids (one with URA
selection marker and one with TRP), 2 .mu.g of GPR119 (human or
mouse receptor) in yeast expression vector (2 .mu.g origin of
replication) and a lithium acetate/polyethylene glycol/TE buffer
was pipetted into an Eppendorf tube. The yeast expression plasmid
containing the receptor/no receptor control has a LEU marker. Yeast
cells were inoculated into this mixture and the reaction proceeds
at 30.degree. C. for 60 min. The yeast cells were then heat-shocked
at 42.degree. C. for 15 min. The cells were then washed and spread
on selection plates. The selection plates are synthetic defined
yeast media minus LEU, URA and TRP (SD-LUT). After incubating at
30.degree. C. for 2-3 days, colonies that grow on the selection
plates were then tested in the LacZ assay.
[0337] In order to perform fluorimetric enzyme assays for
.beta.-galactosidase, yeast cells carrying the human or mouse
GPR119 receptor were grown overnight in liquid SD-LUT medium to an
unsaturated concentration (i.e. the cells were still dividing and
had not yet reached stationary phase). They were diluted in fresh
medium to an optimal assay concentration and 90 .mu.L of yeast
cells added to 96-well black polystyrene plates (Costar).
Compounds, dissolved in DMSO and diluted in a 10% DMSO solution to
10.times. concentration, were added to the plates and the plates
placed at 30.degree. C. for 4 h. After 4 h, the substrate for the
.beta.-galactosidase was added to each well. In these experiments,
Fluorescein di (.beta.-D-galactopyranoside) was used (FDG), a
substrate for the enzyme that releases fluorescein, allowing a
fluorimetric read-out. 20 .mu.L per well of 500 .mu.M FDG/2.5%
Triton X100 was added (the detergent was necessary to render the
cells permeable). After incubation of the cells with the substrate
for 60 min, 20 .mu.L per well of 1M sodium carbonate was added to
terminate the reaction and enhance the fluorescent signal. The
plates were then read in a fluorimeter at 485/535 nm.
[0338] All of Examples 1 to 29 showed activity in this assay giving
an increase in fluorescent signal of at least .about.1.5-fold that
of the background signal (i.e. the signal obtained in the presence
of 1% DMSO without compound). Compounds of the invention which give
an increase of at least 5-fold may be preferred.
cAMP Assay
[0339] A stable cell line expressing recombinant human GPR119 was
established and this cell line was used to investigate the effect
of compounds of the invention on intracellular levels of cyclic AMP
(cAMP). The cell monolayers were washed with phosphate buffered
saline and stimulated at 37.degree. C. for 30 min with various
concentrations of compound in stimulation buffer plus 1% DMSO.
Cells were then lysed and cAMP content determined using the Perkin
Elmer AlphaScreen.TM. (Amplified Luminescent Proximity Homogeneous
Assay) cAMP kit. Buffers and assay conditions were as described in
the manufacturer's protocol.
[0340] Compounds of the invention produced a
concentration-dependent increase in intracellular cAMP level and
generally had an EC.sub.50 of <10 .mu.M. Compounds showing and
EC.sub.50 of less than 1 .mu.M in the cAMP assay may be
preferred.
DPP-IV Assay Method
[0341] DPP-IV activity was measured by monitoring the cleavage of
the fluorogenic peptide substrate,
H-Gly-Pro-7-amino-4-methylcoumarin (GP-AMC) whereby the product
7-amino-4-methylcoumarin is quantified by fluorescence at
excitation 380 nm and emission 460 nm. Assays were carried out in
96-well plates (Black OptiPlate-96F) in a total volume of 100 .mu.L
per well consisting of 50 mM Tris pH 7.6, 100 .mu.M GP-AMC, 10-25
.mu.U recombinant human DPP-IV and a range of inhibitor dilutions
in a final concentration of 1% DMSO. Plates were read in a
fluorimeter after 30 min incubation at 37.degree. C. Recombinant
human DPP-IV residues Asn29-Pro766 was purchased from BioMol.
[0342] All of Examples 1 to 53 showed activity in this assay having
an IC.sub.50 of <20 .mu.M. Compounds of the invention of formula
(Ia) generally have an IC.sub.50 of <20 .mu.M.
Anti-Diabetic Effects of Compounds of the Invention in an In-Vitro
Model of Pancreatic Beta Cells (HIT-T15)
Cell Culture
[0343] HIT-T15 cells (passage 60) were obtained from ATCC, and were
cultured in RPMI1640 medium supplemented with 10% fetal calf serum
and 30 nM sodium selenite. All experiments were done with cells at
less than passage 70, in accordance with the literature, which
describes altered properties of this cell line at passage numbers
above 81 (Zhang H J, Walseth T F, Robertson R P. Insulin secretion
and cAMP metabolism in HIT cells. Reciprocal and serial
passage-dependent relationships. Diabetes. 1989 January;
38(1):44-8).
cAMP Assay
[0344] HIT-T15 cells were plated in standard culture medium in
96-well plates at 100,000 cells/0.1 mL/well and cultured for 24 h
and the medium was then discarded. Cells were incubated for 15 min
at room temperature with 100111 stimulation buffer (Hanks buffered
salt solution, 5 mM HEPES, 0.5 mM IBMX, 0.1% BSA, pH 7.4). This was
discarded and replaced with compound dilutions over the range
0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30 .mu.M in
stimulation buffer in the presence of 0.5% DMSO. Cells were
incubated at room temperature for 30 min. Then 75 uL lysis buffer
(5 mM HEPES, 0.3% Tween-20, 0.1% BSA, pH 7.4) was added per well
and the plate was shaken at 900 rpm for 20 min. Particulate matter
was removed by centrifugation at 3000 rpm for 5 min, then the
samples were transferred in duplicate to 384-well plates, and
processed following the Perkin Elmer AlphaScreen cAMP assay kit
instructions. Briefly 25 .mu.L reactions were set up containing 8
.mu.L sample, 5 .mu.L acceptor bead mix and 12 .mu.L detection mix,
such that the concentration of the final reaction components is the
same as stated in the kit instructions. Reactions were incubated at
room temperature for 150 min, and the plate was read using a
Packard Fusion instrument. Measurements for cAMP were compared to a
standard curve of known cAMP amounts (0.01, 0.03, 0.1, 0.3, 1, 3,
10, 30, 100, 300, 1000 nM) to convert the readings to absolute cAMP
amounts. Data was analysed using XLfit 3 software.
[0345] Representative compounds of the invention were found to
increase cAMP at an EC.sub.K of less than 10 .mu.M. Compounds
showing an EC.sub.50 of less than 1 .mu.M in the cAMP assay may be
preferred.
Insulin Secretion Assay
[0346] HIT-T15 cells are plated in standard culture medium in
12-well plates at 106 cells/1 ml/well and cultured for 3 days and
the medium then discarded. Cells are washed .times.2 with
supplemented Krebs-Ringer buffer (KRB) containing 119 mM NaCl, 4.74
mM KCl, 2.54 mM CaCl.sub.2, 1.19 mM MgSO.sub.4, 1.19 mM
KH.sub.2PO.sub.4, 25 mM NaHCO.sub.3, 10 mM HEPES at pH 7.4 and 0.1%
bovine serum albumin. Cells are incubated with 1 ml KRB at
37.degree. C. for 30 min which is then discarded. This is followed
by a second incubation with KRB for 30 min, which is collected and
used to measure basal insulin secretion levels for each well.
Compound dilutions (0, 0.1, 0.3, 1, 3, 10 .mu.M) are then added to
duplicate wells in 1 ml KRB, supplemented with 5.6 mM glucose.
After 30 min incubation at 37.degree. C. samples are removed for
determination of insulin levels. Measurement of insulin was done
using the Mercodia Rat insulin ELISA kit, following the
manufacturers' instructions, with a standard curve of known insulin
concentrations. For each well, insulin levels are corrected by
subtraction of the basal secretion level from the pre-incubation in
the absence of glucose. Data is analysed using XLfit 3
software.
[0347] Compounds of the invention preferably increase insulin
secretion at an EC.sub.K of less than 10 .mu.M.
Oral Glucose Tolerance Tests
[0348] The effects of compounds of the invention on oral glucose
(Glc) tolerance may be evaluated in male Sprague-Dawley rats. Food
is withdrawn 16 h before administration of Glc and remains
withdrawn throughout the study. Rats have free access to water
during the study. A cut is made to the animals' tails, then blood
(1 drop) is removed for measurement of basal Glc levels 60 min
before administration of the Glc load. Then, the rats are weighed
and dosed orally with test compound or vehicle (20% aqueous
hydroxypropyl-.beta.-cyclodextrin) 45 min before the removal of an
additional blood sample and treatment with the Glc load (2 g
kg.sup.-1 p.o.). Blood samples are taken from the cut tip of the
tail 5, 15, 30, 60, 120, and 180 min after Glc administration.
Blood glucose levels are measured just after collection using a
commercially available glucose-meter (OneTouch.RTM. Ultra.TM. from
Lifescan). Compounds of the invention preferably statistically
reduce the Glc excursion at doses .ltoreq.100 mg kg.sup.-1.
[0349] The effects of compounds of the invention on oral glucose
(Glc) tolerance were evaluated in male C57Bl/6 or male ob/ob mice.
Food was withdrawn 5 h before administration of Glc and remained
withdrawn throughout the study. Mice had free access to water
during the study. A cut was made to the animals' tails, then blood
(20 .mu.L) was removed for measurement of basal Glc levels 45 min
before administration of the Glc load. Then, the mice were weighed
and dosed orally with test compound or vehicle (20% aqueous
hydroxypropyl-.beta.-cyclodextrin or 25% aqueous Gelucire 44/14) 30
min before the removal of an additional blood sample (20 .mu.L) and
treatment with the Glc load (2-5 g kg.sup.-1 p.o.). Blood samples
(20 .mu.L) were then taken 25, 50, 80, 120, and 180 min after Glc
administration. The 20 .mu.L blood samples for measurement of Glc
levels were taken from the cut tip of the tail into disposable
micro-pipettes (Dade Diagnostics Inc., Puerto Rico) and the sample
added to 480 .mu.L of haemolysis reagent. Duplicate 20 .mu.L
aliquots of the diluted haemolysed blood were then added to 180
.mu.L of Trinders glucose reagent (Sigma enzymatic (Trinder)
colorimetric method) in a 96-well assay plate. After mixing, the
samples were left at room temperature for 30 min before being read
against Glc standards (Sigma glucose/urea nitrogen combined
standard set). Compounds of the invention statistically reduced the
Glc excursion at doses .ltoreq.100 mg kg.sup.-1, for example at a
dose of 30 mg kg.sup.-1 the compound of Example 18 showed a >40%
reduction in the Glc excursion.
* * * * *